Human antibodies that bind human il-12 and methods for producing

ABSTRACT

Human antibodies, preferably recombinant human antibodies, that specifically bind to human interleukin-12 (hIL-12) are disclosed. Preferred antibodies have high affinity for hIL-12 and neutralize hIL-12 activity in vitro and in vivo. An antibody of the invention can be a full-length antibody or an antigen-binding portion thereof. The antibodies, or antibody portions, of the invention are useful for detecting hIL-12 and for inhibiting hIL-12 activity, e.g., in a human subject suffering from a disorder in which hIL-12 activity is detrimental. Nucleic acids, vectors and host cells for expressing the recombinant human antibodies of the invention, and methods of synthesizing the recombinant human antibodies, are also encompassed by the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/418,049, filed Mar. 12, 2012, which is a continuation of U.S. patentapplication Ser. No. 12/121,615, filed May 15, 2008, now abandoned,which is a divisional of U.S. patent application Ser. No. 10/884,830,filed Jul. 1, 2004, now U.S. Pat. No. 7,504,485, which is a divisionalof U.S. patent application Ser. No. 09/534,717, filed Mar. 24, 2000, nowU.S. Pat. No. 6,914,128, which is a non-provisional application claimingpriority to U.S. Provisional Application Ser. No. 60/126,603, filed Mar.25, 1999. The entire contents of each of the foregoing applications arehereby incorporated by reference.

BACKGROUND OF THE INVENTION

Human interleukin 12 (IL-12) has recently been characterized as acytokine with a unique structure and pleiotropic effects (Kobayashi, etal. (1989) J. Exp Med. 170:827-845; Seder, et al. (1993) Proc. Natl.Acad. Sci. 90:10188-10192; Ling, et al. (1995) J. Exp Med. 154:116-127;Podlaski, et al. (1992) Arch. Biochem. Biophys. 294:230-237). IL-12plays a critical role in the pathology associated with several diseasesinvolving immune and inflammatory responses. A review of IL-12, itsbiological activities, and its role in disease can be found in Gately etal. (1998) Ann. Rev. Immunol. 16: 495-521.

Structurally, IL-12 is a heterodimeric protein comprising a 35 kDasubunit (p35) and a 40 kDa subunit (p40) which are both linked togetherby a disulfide bridge (referred to as the “p70 subunit”). Theheterodimeric protein is produced primarily by antigen-presenting cellssuch as monocytes, macrophages and dendritic cells. These cell typesalso secrete an excess of the p40 subunit relative to p70 subunit. Thep40 and p35 subunits are genetically unrelated and neither has beenreported to possess biological activity, although the p40 homodimer mayfunction as an IL-12 antagonist.

Functionally, IL-12 plays a central role in regulating the balancebetween antigen specific T helper type (Th1) and type 2 (Th2)lymphocytes. The Th1 and Th2 cells govern the initiation and progressionof autoimmune disorders, and IL-12 is critical in the regulation ofTh₁-lymphocyte differentiation and maturation. Cytokines released by theTh1 cells are inflammatory and include interferon γ (IFNγ), IL-2 andlymphotoxin (LT). Th2 cells secrete IL-4, IL-5, IL-6, IL-10 and IL-13 tofacilitate humoral immunity, allergic reactions, and immunosuppression.

Consistent with the preponderance of Th1 responses in autoimmunediseases and the proinflammatory activities of IFNγ, IL-12 may play amajor role in the pathology associated with many autoimmune andinflammatory diseases such as rheumatoid arthritis (RA), multiplesclerosis (MS), and Crohn's disease.

Human patients with MS have demonstrated an increase in IL-12 expressionas documented by p40 mRNA levels in acute MS plaques. (Windhagen et al.,(1995) J. Exp. Med. 182: 1985-1996). In addition, ex vivo stimulation ofantigen-presenting cells with CD40L-expressing T cells from MS patientsresulted in increased IL-12 production compared with control T cells,consistent with the observation that CD40/CD40L interactions are potentinducers of IL-12.

Elevated levels of IL-12 p70 have been detected in the synovia of RApatients compared with healthy controls (Morita et al (1998) Arthritisand Rheumatism. 41: 306-314). Cytokine messenger ribonucleic acid (mRNA)expression profile in the RA synovia identified predominantly Th1cytokines. (Bucht et al., (1996) Clin. Exp. Immunol. 103: 347-367).IL-12 also appears to play a critical role in the pathology associatedwith Crohn's disease (CD). Increased expression of INFγ and IL-12 hasbeen observed in the intestinal mucosa of patients with this disease(Fais et al. (1994) J. Interferon Res. 14:235-238; Parronchi et al.,(1997) Am. J. Path. 150:823-832; Monteleone et al., (1997)Gastroenterology. 112:1169-1178, and Berrebi et al., (1998) Am. J. Path152:667-672). The cytokine secretion profile of T cells from the laminapropria of CD patients is characteristic of a predominantly Th1response, including greatly elevated IFNγ levels (Fuss, et al., (1996)J. Immunol. 157:1261-1270). Moreover, colon tissue sections from CDpatients show an abundance of IL-12 expressing macrophages and IFNγexpressing T cells (Parronchi et al (1997) Am. J. Path. 150:823-832).

Due to the role of human IL-12 in a variety of human disorders,therapeutic strategies have been designed to inhibit or counteract IL-12activity. In particular, antibodies that bind to, and neutralize, IL-12have been sought as a means to inhibit IL-12 activity. Some of theearliest antibodies were murine monoclonal antibodies (mAbs), secretedby hybridomas prepared from lymphocytes of mice immunized with IL-12(see e.g., World Patent Application Publication No. WO 97/15327 byStrober et al.; Neurath et al. (1995) J. Exp. Med. 182:1281-1290;Duchmann et al. (1996) J. Immunol. 26:934-938). These murine IL-12antibodies are limited for their use in vivo due to problems associatedwith administration of mouse antibodies to humans, such as short serumhalf life, an inability to trigger certain human effector functions andelicitation of an unwanted immune response against the mouse antibody ina human (the “human anti-mouse antibody” (HAMA) reaction).

In general, attempts to overcome the problems associated with use offully-murine antibodies in humans, have involved genetically engineeringthe antibodies to be more “human-like.” For example, chimericantibodies, in which the variable regions of the antibody chains aremurine-derived and the constant regions of the antibody chains arehuman-derived, have been prepared (Junghans, et al. (1990) Cancer Res.50:1495-1502; Brown et al. (1991) Proc. Natl. Acad. Sci. 88:2663-2667;Kettleborough et al. (1991) Protein Engineering. 4:773-783). However,because these chimeric and humanized antibodies still retain some murinesequences, they still may elicit an unwanted immune reaction, the humananti-chimeric antibody (HACA) reaction, especially when administered forprolonged periods.

A preferred IL-12 inhibitory agent to murine antibodies or derivativesthereof (e.g., chimeric or humanized antibodies) would be an entirelyhuman anti-IL-12 antibody, since such an agent should not elicit theHAMA reaction, even if used for prolonged periods. However, suchantibodies have not been described in the art and, therefore are stillneeded.

SUMMARY OF THE INVENTION

The present invention provides human antibodies that bind human IL-12.The invention also relates to the treatment or prevention of acute orchronic diseases or conditions whose pathology involves IL-12, using thehuman anti-IL-12 antibodies of the invention.

In one aspect, the invention provides an isolated human antibody, or anantigen-binding portion thereof, that binds to human IL-12.

In one embodiment, the invention provides a selectively mutated humanIL-12 antibody, comprising:

a human antibody or antigen-binding portion thereof, selectively mutatedat a preferred selective mutagenesis position, contact or hypermutationposition with an activity enhancing amino acid residue such that itbinds to human IL-12.

In a preferred embodiment, the invention provides a selectively mutatedhuman IL-12 antibody, comprising:

a human antibody or antigen-binding portion thereof, selectively mutatedat a preferred selective mutagenesis position with an activity enhancingamino acid residue such that it binds to human IL-12.

In another preferred embodiment, the selectively mutated human IL-12antibody or antigen-binding portion thereof is selectively mutated atmore than one preferred selective mutagenesis position, contact orhypermutation positions with an activity enhancing amino acid residue.In another preferred embodiment, the selectively mutated human IL-12antibody or antigen-binding portion thereof is selectively mutated at nomore than three preferred selective mutagenesis positions, contact orhypermutation positions. In another preferred embodiment, theselectively mutated human IL-12 antibody or antigen-binding portionthereof is selectively mutated at no more than two preferred selectivemutagenesis position, contact or hypermutation positions. In yet anotherpreferred embodiment, the selectively mutated human IL-12 antibody orantigen-binding portion thereof, is selectively mutated such that atarget specificity affinity level is attained, the target level beingimproved over that attainable when selecting for an antibody against thesame antigen using phage display technology. In another preferredembodiment, the selectively mutated human IL-12 antibody further retainsat least one desirable property or characteristic, e.g., preservation ofnon-cross reactivity with other proteins or human tissues, preservationof epitope recognition, production of an antibody with a close to agermline immunoglobulin sequence.

In another embodiment, the invention provides an isolated humanantibody, or antigen-binding portion thereof, that binds to human IL-12and dissociates from human IL-12 with a K_(off) rate constant of 0.1 s⁻¹or less, as determined by surface plasmon resonance, or which inhibitsphytohemagglutinin blast proliferation in an in vitro phytohemagglutininblast proliferation assay (PHA assay) with an IC₅₀ of 1×10⁻⁶ M or less.More preferably, the isolated human antibody or an antigen-bindingportion thereof, dissociates from human IL-12 with a K_(off) rateconstant of 1×10⁻² s⁻¹ or less, or inhibits phytohemagglutinin blastproliferation in an in vitro PHA assay with an IC₅₀ of 1×10⁻⁷ M or less.More preferably, the isolated human antibody, or an antigen-bindingportion thereof, dissociates from human IL-12 with a K_(off) rateconstant of 1×10⁻³ s⁻¹ or less, or inhibits phytohemagglutinin blastproliferation in an in vitro PHA assay with an IC₅₀ of 1×10⁻⁸ M or less.More preferably, the isolated human antibody, or an antigen-bindingportion thereof, dissociates from human IL-12 with a K_(off) rateconstant of 1×10⁻⁴ s⁻¹ or less, or inhibits phytohemagglutinin blastproliferation in an in vitro PHA assay with an IC₅₀ of 1×10⁻⁹ M or less.More preferably, the isolated human antibody, or an antigen-bindingportion thereof, dissociates from human IL-12 with a K_(off) rateconstant of 1×10⁻⁵ s⁻¹ or less, or inhibits phytohemagglutinin blastproliferation in an in vitro PHA assay with an IC₅₀ of 1×10⁻¹⁰ M orless. Even more preferably, the isolated human antibody, or anantigen-binding portion thereof, dissociates from human IL-12 with aK_(off) rate constant of 1×10⁻⁵ s⁻¹ or less, or inhibitsphytohemagglutinin blast proliferation in an in vitro PHA assay with anIC₅₀ of 1×10⁻¹¹ M or less.

In another embodiment, the invention provides an isolated humanantibody, or an antigen-binding portion thereof, which has the followingcharacteristics:

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁶ M or less;

b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ IDNO: 1; and

c) has a light chain CDR3 comprising the amino acid sequence of SEQ IDNO: 2.

In a preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, has a heavy chain CDR2 comprising theamino acid sequence of SEQ ID NO: 3; and has a light chain CDR2comprising the amino acid sequence of SEQ ID NO: 4. In a preferredembodiment, the isolated human antibody, or an antigen-binding portionthereof, has a heavy chain CDR1 comprising the amino acid sequence ofSEQ ID NO: 5; and has a light chain CDR1 comprising the amino acidsequence of SEQ ID NO: 6. In a preferred embodiment, the isolated humanantibody, or antigen binding portion thereof, has a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 7; and has alight chain variable region comprising the amino acid sequence of SEQ IDNO: 8.

In another embodiment, the invention provides an isolated humanantibody, or an antigen-binding portion thereof, which has the followingcharacteristics:

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁹ M or less;

b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ IDNO: 9; and

c) has a light chain CDR3 comprising the amino acid sequence of SEQ IDNO: 10.

In a preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, has a heavy chain CDR2 comprising theamino acid sequence of SEQ ID NO: 11; and has a light chain CDR2comprising the amino acid sequence of SEQ ID NO: 12. In a preferredembodiment, the isolated human antibody, or an antigen-binding portionthereof, has a heavy chain CDR1 comprising the amino acid sequence ofSEQ ID NO: 13; and has a light chain CDR1 comprising the amino acidsequence of SEQ ID NO: 14. In a preferred embodiment, the isolated humanantibody has a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 15; and has a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 16.

In another embodiment, the invention provides an isolated humanantibody, or an antigen-binding portion thereof, which

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁹ M or less;

b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ IDNO: 17; and

c) has a light chain CDR3 comprising the amino acid sequence of SEQ IDNO: 18.

In a preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, has a heavy chain CDR2 comprising theamino acid sequence of SEQ ID NO: 19; and a light chain CDR2 comprisingthe amino acid sequence of SEQ ID NO: 20. In a preferred embodiment, theisolated human antibody, or an antigen-binding portion thereof, has aheavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21;and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:22. In a preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, has the heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 23, and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 24. Ina preferred embodiment, the isolated human antibody comprises a heavychain constant region selected from the group consisting of IgG1, IgG2,IgG3, IgG4, IgM, IgA and IgE constant regions or any allelic variationthereof as discussed in Kabat et al. (Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242),included herein by reference. In a more preferred embodiment, theantibody heavy chain constant region is IgG1. In another preferredembodiment, the isolated human antibody is a Fab fragment, or a F(ab′)₂fragment or a single chain Fv fragment.

In another embodiment, the invention provides an isolated humanantibody, or an antigen-binding portion thereof, which

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁹ M or less;

b) has a heavy chain CDR3 comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NO: 404-SEQ ID NO: 469; and

c) has a light chain CDR3 comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NO: 534-SEQ ID NO: 579.

In a preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, has a heavy chain CDR2 comprising theamino acid sequence selected from the group consisting of SEQ IDNO:335-SEQ ID NO: 403; and a light chain CDR2 comprising the amino acidsequence selected from the group consisting of SEQ ID NO: 506-SEQ ID NO:533. In a preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, has a heavy chain CDR1 comprising theamino acid sequence selected from the group consisting of SEQ ID NO:288-SEQ ID NO: 334; and a light chain CDR1 comprising the amino acidsequence selected from the group consisting of SEQ ID NO: 470-SEQ ID NO:505. In a preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, comprising a the heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 23, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:24. In a preferred embodiment, the isolated human antibody comprises aheavy chain constant region, or an Fab fragment or a F(ab′)₂ fragment ora single chain Fv fragment as described above.

In another embodiment, the invention provides an isolated humanantibody, or an antigen-binding portion thereof, which

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁹ M or less;

b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ IDNO: 25; and

c) has a light chain CDR3 comprising the amino acid sequence of SEQ IDNO: 26.

In a preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, has a heavy chain CDR2 comprising theamino acid sequence of SEQ ID NO: 27; and a light chain CDR2 comprisingthe amino acid sequence of SEQ ID NO: 28. In a preferred embodiment, theisolated human antibody, or an antigen-binding portion thereof, has aheavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29;and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:30. In a preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, which has a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 31, and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 32. Ina preferred embodiment, the isolated human antibody comprises a heavychain constant region, or an Fab fragment, or a F(ab′)₂ fragment or asingle chain Fv fragment as described above.

In another embodiment, the invention provides an isolated humanantibody, or an antigen-binding portion thereof, which

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁶ M or less;

b) comprises a heavy chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 3 and a heavy chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 5, or a mutant thereof having one or more amino acidsubstitutions at a contact position or a hypermutation position, whereinsaid mutant has a k_(off) rate no more than 10-fold higher than theantibody comprising a heavy chain CDR3 comprising the amino acidsequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acidsequence of SEQ ID NO: 3, and a heavy chain CDR1 comprising the aminoacid sequence of SEQ ID NO: 5; and

c) comprises a light chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 2, a light chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 4, and a light chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 6, or a mutant thereof having one or more amino acidsubstitutions at a contact position or a hypermutation position, whereinsaid mutant has a k_(off) rate no more than 10-fold higher than theantibody comprising a light chain CDR3 comprising the amino acidsequence of SEQ ID NO: 2, a light chain CDR2 comprising the amino acidsequence of SEQ ID NO: 4, and a light chain CDR1 comprising the aminoacid sequence of SEQ ID NO: 6.

In another embodiment, the invention provides an isolated humanantibody, or an antigen-binding portion thereof, which

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁹ M or less;

b) comprises a heavy chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 11 and a heavy chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 13, or a mutant thereof having one or more amino acidsubstitutions at a contact position or a hypermutation position, whereinsaid mutant has a k_(off) rate no more than 10-fold higher than theantibody comprising a heavy chain CDR3 comprising the amino acidsequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acidsequence of SEQ ID NO: 11, and a heavy chain CDR1 comprising the aminoacid sequence of SEQ ID NO: 13; and

c) comprises a light chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 12, and a light chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 14, or a mutant thereof having one or more amino acidsubstitutions at a preferred selective mutagenesis position, contactposition or a hypermutation position, wherein said mutant has a k_(off)rate no more than 10-fold higher than the antibody comprising a lightchain CDR3 comprising the amino acid sequence of SEQ ID NO: 10, a lightchain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and alight chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14.

In another embodiment, the invention provides an isolated humanantibody, or an antigen-binding portion thereof, which

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁹ M or less;

b) comprises a heavy chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 19 and a heavy chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 21, or a mutant thereof having one or more amino acidsubstitutions at a preferred selective mutagenesis position, contactposition or a hypermutation position, wherein said mutant has a k_(off)rate no more than 10-fold higher than the antibody comprising a heavychain CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a heavychain CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and aheavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21;and

c) comprises a light chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 18, a light chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 20, and a light chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 22, or a mutant thereof having one or more amino acidsubstitutions at preferred selective mutagenesis position, contactposition or a hypermutation position, wherein said mutant has a k_(off)rate no more than 10-fold higher than the antibody comprising a lightchain CDR3 comprising the amino acid sequence of SEQ ID NO: 18, a lightchain CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and alight chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22.

The invention also provides nucleic acid molecules encoding antibodies,or antigen binding portions thereof, of the invention. A preferredisolated nucleic acid encodes the heavy chain CDR3 comprising the aminoacid sequence of SEQ ID NO: 17. The isolated nucleic acid encoding anantibody heavy chain variable region. In another embodiment, theisolated nucleic acid encodes the CDR2 of the antibody heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 19. Inanother embodiment, the isolated nucleic acid encodes the CDR1 of theantibody heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 21. In another embodiment, the isolated nucleic acidencodes an antibody heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 23. In another embodiment, the isolatednucleic acid encodes the light chain CDR3 comprising the amino acidsequence of SEQ ID NO: 18. The isolated nucleic acid encoding anantibody light chain variable region. In another embodiment, theisolated nucleic acid encodes the CDR2 of the antibody light chainvariable region comprising the amino acid sequence of SEQ ID NO: 20. Inanother embodiment, the isolated nucleic acid encodes the CDR1 of theantibody light chain variable region comprising the amino acid sequenceof SEQ ID NO: 22. In another embodiment, the isolated nucleic acidencodes an antibody light chain variable region comprising the aminoacid sequence of SEQ ID NO: 24.

In another embodiment, the invention provides an isolated humanantibody, or an antigen-binding portion thereof, which

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁹ M or less;

b) comprises a heavy chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 27 and a heavy chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 29, or a mutant thereof having one or more amino acidsubstitutions at a preferred selective mutagenesis position, contactposition or a hypermutation position, wherein said mutant has a k_(off)rate no more than 10-fold higher than the antibody comprising a heavychain CDR3 comprising the amino acid sequence of SEQ ID NO: 25, a heavychain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and aheavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29;and

c) comprises a light chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 28, and a light chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 30, or a mutant thereof having one or more amino acidsubstitutions at a preferred selective mutagenesis position, contactposition or a hypermutation position, wherein said mutant has a k_(off)rate no more than 10-fold higher than the antibody comprising a lightchain CDR3 comprising the amino acid sequence of SEQ ID NO: 26, a lightchain CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and alight chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30.

A preferred isolated nucleic acid encodes the heavy chain CDR3comprising the amino acid sequence of SEQ ID NO: 25. The isolatednucleic acid encoding an antibody heavy chain variable region. Inanother embodiment, the isolated nucleic acid encodes the CDR2 of theantibody heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 27. In another embodiment, the isolated nucleic acidencodes the CDR1 of the antibody heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 29. In another embodiment, theisolated nucleic acid encodes an antibody heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 31. In anotherembodiment, the isolated nucleic acid encodes the light chain CDR3comprising the amino acid sequence of SEQ ID NO: 26. The isolatednucleic acid encoding an antibody light chain variable region. Inanother embodiment, the isolated nucleic acid encodes the CDR2 of theantibody light chain variable region comprising the amino acid sequenceof SEQ ID NO: 28. In another embodiment, the isolated nucleic acidencodes the CDR1 of the antibody light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 30. In another embodiment, theisolated nucleic acid encodes an antibody light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 32.

In another aspect, the invention provides an isolated human antibody, oran antigen-binding portion thereof, which has the followingcharacteristics:

-   -   a) that binds to human IL-12 and dissociates from human IL-12        with a k_(off) rate constant of 0.1 s⁻¹ or less, as determined        by surface plasmon resonance, or which inhibits        phytohemagglutinin blast proliferation in an in vitro        phytohemagglutinin blast proliferation assay (PHA assay) with an        IC₅₀ of 1×10⁻⁶ M or less.    -   b) has a heavy chain variable region comprising an amino acid        sequence selected from a member of the V_(H)3 germline family,        wherein the heavy chain variable region has a mutation at a        preferred selective mutagenesis position, contact or        hypermutation position with an activity enhancing amino acid        residue.    -   c) has a light chain variable region comprising an amino acid        sequence selected from a member of the V_(λ)1 germline family,        wherein the light chain variable region has a mutation at a        preferred selective mutagenesis position, contact position or        hypermutation position with an activity enhancing amino acid        residue.

In another embodiment, the invention provides an isolated humanantibody, or an antigen-binding portion thereof, which has the followingcharacteristics:

-   -   a) that binds to human IL-12 and dissociates from human IL-12        with a k_(off) rate constant of 0.1 s⁻¹ or less, as determined        by surface plasmon resonance, or which inhibits        phytohemagglutinin blast proliferation in an in vitro        phytohemagglutinin blast proliferation assay (PHA assay) with an        IC₅₀ of 1×10⁻⁶ M or less.    -   b) has a heavy chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:        595-667, wherein the heavy chain variable region has a mutation        at a preferred selective mutagenesis position, contact position        or hypermutation position with an activity enhancing amino acid        residue.    -   c) has a light chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:        669-675, wherein the light chain variable region has a mutation        at a preferred selective mutagenesis position, contact or        hypermutation position with an activity enhancing amino acid        residue.

In another embodiment, the invention provides an isolated humanantibody, or an antigen-binding portion thereof, which has the followingcharacteristics:

-   -   a) that binds to human IL-12 and dissociates from human IL-12        with a k_(off) rate constant of 0.1 s⁻¹ or less, as determined        by surface plasmon resonance, or which inhibits        phytohemagglutinin blast proliferation in an in vitro        phytohemagglutinin blast proliferation assay (PHA assay) with an        IC₅₀ of 1×10⁻⁶ M or less.    -   b) has a heavy chain variable region comprising the COS-3        germline amino acid sequence, wherein the heavy chain variable        region has a mutation at a preferred selective mutagenesis        position, contact or hypermutation position with an activity        enhancing amino acid residue.    -   c) has a light chain variable region comprising the DPL8        germline amino acid sequence, wherein the light chain variable        region has a mutation at a preferred selective mutagenesis        position, contact or hypermutation position with an activity        enhancing amino acid residue.

In another embodiment, the invention provides an isolated humanantibody, or an antigen-binding portion thereof, which has the followingcharacteristics:

-   -   a) that binds to human IL-12 and dissociates from human IL-12        with a k_(off) rate constant of 0.1 s⁻¹ or less, as determined        by surface plasmon resonance, or which inhibits        phytohemagglutinin blast proliferation in an in vitro        phytohemagglutinin blast proliferation assay (PHA assay) with an        IC₅₀ of 1×10⁻⁶ M or less.    -   b) has a heavy chain variable region comprising an amino acid        sequence selected from a member of the V_(H)3 germline family,        wherein the heavy chain variable region comprises a CDR2 that is        structurally similar to CDR2s from other V_(H)3 germline family        members, and a CDR1 that is structurally similar to CDR1s from        other V_(H)3 germline family members, and wherein the heavy        chain variable region has a mutation at a preferred selective        mutagenesis position, contact or hypermutation position with an        activity enhancing amino acid residue;    -   c) has a light chain variable region comprising an amino acid        sequence selected from a member of the V_(λ)1 germline family,        wherein the light chain variable region comprises a CDR2 that is        structurally similar to CDR2s from other V_(λ)1 germline family        members, and a CDR1 that is structurally similar to CDR1s from        other V_(λ)1 germline family members, and wherein the light        chain variable region has a mutation at a preferred selective        mutagenesis position, contact or hypermutation position with an        activity enhancing amino acid residue.

In a preferred embodiment, the isolated human antibody, or antigenbinding portion thereof, has a mutation in the heavy chain CDR3. Inanother preferred embodiment, the isolated human antibody, or antigenbinding portion thereof, has a mutation in the light chain CDR3. Inanother embodiment, the isolated human antibody, or antigen bindingportion thereof, has a mutation in the heavy chain CDR2. In anotherpreferred embodiment, the isolated human antibody, or antigen bindingportion thereof, has a mutation in the light chain CDR2. In anotherpreferred embodiment, the isolated human antibody, or antigen bindingportion thereof, has a mutation in the heavy chain CDR1. In anotherpreferred embodiment, the isolated human antibody, or antigen bindingportion thereof, has a mutation in the light chain CDR1.

In another aspect, the invention provides recombinant expression vectorscarrying the antibody-encoding nucleic acids of the invention, and hostcells into which such vectors have been introduced, are also encompassedby the invention, as are methods of making the antibodies of theinvention by culturing the host cells of the invention.

In another aspect, the invention provides an isolated human antibody, orantigen-binding portion thereof, that neutralizes the activity of humanIL-12, and at least one additional primate IL-12 selected from the groupconsisting of baboon IL-12, marmoset IL-12, chimpanzee IL-12, cynomolgusIL-12 and rhesus IL-12, but which does not neutralize the activity ofthe mouse IL-12.

In another aspect, the invention provides a pharmaceutical compositioncomprising the antibody or an antigen binding portion thereof, of theinvention and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a composition comprising theantibody or an antigen binding portion thereof, and an additional agent,for example, a therapeutic agent.

In another aspect, the invention provides a method for inhibiting humanIL-12 activity comprising contacting human IL-12 with the antibody ofthe invention, e.g., J695, such that human IL-12 activity is inhibited.

In another aspect, the invention provides a method for inhibiting humanIL-12 activity in a human subject suffering from a disorder in whichIL-12 activity is detrimental, comprising administering to the humansubject the antibody of the invention, e.g., J695, such that human IL-12activity in the human subject is inhibited. The disorder can be, forexample, Crohn's disease, multiple sclerosis or rheumatoid arthritis.

In another aspect, the invention features a method for improving theactivity of an antibody, or an antigen binding portion thereof, toattain a predetermined target activity, comprising:

a) providing a parent antibody a antigen-binding portion thereof;

b) selecting a preferred selective mutagenesis position selected fromgroup consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,L32, L50, L91, L92, L93, L94.

c) individually mutating the selected preferred selective mutagenesisposition to at least two other amino acid residues to hereby create afirst panel of mutated antibodies, or antigen binding portions thereof;

d) evaluating the activity of the first panel of mutated antibodies, orantigen binding portions thereof to determined if mutation of a singleselective mutagenesis position produces an antibody or antigen bindingportion thereof with the predetermined target activity or a partialtarget activity;

e) combining in a stepwise fashion, in the parent antibody, or antigenbinding portion thereof, individual mutations shown to have an improvedactivity, to form combination antibodies, or antigen binding portionsthereof.

f) evaluating the activity of the combination antibodies, or antigenbinding portions thereof to determined if the combination antibodies, orantigen binding portions thereof have the predetermined target activityor a partial target activity.

g) if steps d) or f) do not result in an antibody or antigen bindingportion thereof having the predetermined target activity, or result anantibody with only a partial activity, additional amino acid residuesselected from the group consisting of H34, H50, H53, H54, H95, H96, H97,H98, L30A and L96 are mutated to at least two other amino acid residuesto thereby create a second panel of mutated antibodies orantigen-binding portions thereof;

h) evaluating the activity of the second panel of mutated antibodies orantigen binding portions thereof, to determined if mutation of a singleamino acid residue selected from the group consisting of H35, H50, H53,H54, H95, H96, H97, H98, L30A and L96 results an antibody or antigenbinding portion thereof, having the predetermined target activity or apartial activity;

i) combining in stepwise fashion in the parent antibody, orantigen-binding portion thereof, individual mutations of step g) shownto have an improved activity, to form combination antibodies, or antigenbinding portions thereof;

j) evaluating the activity of the combination antibodies or antigenbinding portions thereof, to determined if the combination antibodies,or antigen binding portions thereof have the predetermined targetactivity or a partial target activity;

k) if steps h) or j) do not result in an antibody or antigen bindingportion thereof having the predetermined target activity, or result inan antibody with only a partial activity, additional amino acid residuesselected from the group consisting of H33B, H52B and L31A are mutated toat least two other amino acid residues to thereby create a third panelof mutated antibodies or antigen binding portions thereof;

l) evaluating the activity of the third panel of mutated antibodies orantigen binding portions thereof, to determine if a mutation of a singleamino acid residue selected from the group consisting of H33B, H52B andL31A resulted in an antibody or antigen binding portion thereof, havingthe predetermined target activity or a partial activity;

m) combining in a stepwise fashion in the parent antibody, or antigenbinding portion thereof, individual mutation of step k) shown to have animproved activity, to form combination antibodies, or antigen bindingportions, thereof;

n) evaluating the activity of the combination antibodies orantigen-binding portions thereof, to determine if the combinationantibodies, or antigen binding portions thereof have the predeterminedtarget activity to thereby produce an antibody or antigen bindingportion thereof with a predetermined target activity.

In another aspect, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof;

b) selecting a preferred selective mutagenesis position, contact orhypermutation position within a complementarity determining region (CDR)for mutation, thereby identifying a selected preferred selectivemutagenesis position, contact or hypermutation position;

c) individually mutating said selected preferred selective mutagenesisposition, contact or hypermutation position to at least two other aminoacid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof;

e) repeating steps b) through d) for at least one other contact orhypermutation position;

f) combining, in the parent antibody, or antigen-binding portionthereof, individual mutations shown to have improved activity, to formcombination antibodies, or antigen-binding portions thereof; and

g) evaluating the activity of the combination antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof; until an antibody, or antigen-bindingportion thereof, with an improved activity, relative to the parentantibody, or antigen-binding portion thereof, is obtained.

In one embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a recombinant parent antibody or antigen-binding portionthereof; that was obtained by selection in a phage-display system butwhose activity is not further improved by mutagenesis in saidphage-display system;

b) selecting a preferred selective mutagenesis position, contact orhypermutation position within a complementarity determining region (CDR)for mutation, thereby identifying a selected contact or hypermutationposition;

c) individually mutating said selected preferred selective mutagenesisposition, contact or hypermutation position to at least two other aminoacid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof, and expressing said panel in anon-phage display system;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof;

e) repeating steps b) through d) for at least one other contact orhypermutation position;

f) combining, in the parent antibody, or antigen-binding portionthereof, individual mutations shown to have improved activity, to formcombination antibodies, or antigen-binding portions thereof; and

g) evaluating the activity of the combination antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof; until an antibody, or antigen-bindingportion thereof, with an improved activity, relative to the parentantibody, or antigen-binding portion thereof, is obtained.

In a preferred embodiment, the contact positions are selected from thegroup consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52,L53, L55, L91, L92, L93, L94 and L96. In another preferred embodiment,the hypermutation positions are selected from the group consisting ofH30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93. In amore preferred embodiment the residues for selective mutagenesis areselected from the preferred selective mutagenesis positions from thegroup consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,L32, L50, L91, L92, L93, L94. In a more preferred embodiment, thecontact positions are selected from the group consisting of L50 and L94.

In another embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a recombinant parent antibody or antigen-binding portionthereof;

b) selecting a preferred selective mutagenesis position, contact orhypermutation position within a complementarity determining region (CDR)for mutation, thereby identifying a selected contact or hypermutationposition;

c) individually mutating said selected preferred selective mutagenesisposition, contact or hypermutation position to at least two other aminoacid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof and expressing said panel in anappropriate expression system;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof for at least one other property or characteristics,wherein the property or characteristic is one that needs to be retainedin the antibody;

until an antibody, or antigen-binding portion thereof, with an improvedactivity and at least one retained property or characteristic, relativeto the parent antibody, or antigen-binding portion thereof, is obtained.

In a preferred embodiment, the contact positions are selected from thegroup consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52,L53, L55, L91, L92, L93, L94 and L96 and the other characteristic isselected from 1) preservation of non-crossreactivity with other proteinsor human tissues, 2) preservation of epitope recognition, i.e.recognizing p40 epitope preferably in the context of the p70 p40/p35heterodimer preventing binding interference from free, soluble p40and/or 3) to produce an antibody with a close to germline immunoglobulinsequence. In another preferred embodiment, the hypermutation positionsare selected from the group consisting of H30, H31, H31B, H32, H52, H56,H58, L30, L31, L32, L53 and L93 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence. Ina more preferred embodiment the residues for selective mutagenesis areselected from the preferred selective mutagenesis positions from thegroup consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,L32, L50, L91, L92, L93, L94 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence. Ina more preferred embodiment, the contact positions are selected from thegroup consisting of L50 and L94 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence.

In another embodiment of the invention provides a method for improvingthe activity of an antibody, or antigen-binding portion thereof,comprising:

a) providing a recombinant parent antibody or antigen-binding portionthereof; that was obtained by selection in a phage-display system butwhose activity cannot be further improved by mutagenesis in saidphage-display system;

b) selecting a preferred selective mutagenesis position, contact orhypermutation position within a complementarity determining region (CDR)for mutation, thereby identifying a selected preferred selectivemutagenesis position, contact or hypermutation position;

c) individually mutating said selected preferred selective mutagenesisposition, contact or hypermutation position to at least two other aminoacid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof, and expressing said panel in anon-phage display system;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof for at least one other property or characteristic,wherein the property or characteristic is one that needs to be retained,until an antibody, or antigen-binding portion thereof, with an improvedactivity and at least one retained property or characteristic, relativeto the parent antibody, or antigen-binding portion thereof, is obtained.

f) repeating steps a) through e) for at least one other preferredselective mutagenesis position, contact or hypermutation position;

g) combining, in the parent antibody, or antigen-binding portionthereof, at least two individual activity enhancing amino acid residuesshown to have improved activity and at least on retained property orcharacteristic, to form combination antibodies, or antigen-bindingportions thereof; and

h) evaluating the activity of the combination antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof; until an antibody, or antigen-bindingportion thereof, with an improved activity and at least one retainedproperty or characteristic, relative to the parent antibody, orantigen-binding portion thereof, is obtained.

In a preferred embodiment, the contact positions are selected from thegroup consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52,L53, L55, L91, L92, L93, L94 and L96 and the other characteristic isselected from 1) preservation of non-crossreactivity with other proteinsor human tissues, 2) preservation of epitope recognition, i.e.recognizing p40 epitope preferably in the context of the p70 p40/p35heterodimer preventing binding interference from free, soluble p40and/or 3) to produce an antibody with a close to germline immunoglobulinsequence. In another preferred embodiment, the hypermutation positionsare selected from the group consisting of H30, H31, H31B, H32, H52, H56,H58, L30, L31, L32, L53 and L93 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence. Ina more preferred embodiment the residues for selective mutagenesis areselected from the preferred selective mutagenesis positions from thegroup consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,L32, L50, L91, L92, L93, L94 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence. Ina more preferred embodiment, the contact positions are selected from thegroup consisting of L50 and L94 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence.

In another embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a recombinant parent antibody or antigen-binding portionthereof; that was obtained by selection in a phage-display system butwhose activity cannot be further improved by mutagenesis in saidphage-display system;

b) selecting a contact or hypermutation position within acomplementarity determining region (CDR) for mutation, therebyidentifying a selected contact or hypermutation position;

c) individually mutating said selected contact or hypermutation positionto at least two other amino acid residues to thereby create a panel ofmutated antibodies, or antigen-binding portions thereof, and expressingsaid panel in a non-phage display system;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof for at least one other property or characteristics,wherein the property or characteristic is one that needs to be retained;

until an antibody, or antigen-binding portion thereof, with an improvedactivity and at least one retained property or characteristic, relativeto the parent antibody, or antigen-binding portion thereof, is obtained.

In a preferred embodiment, the contact positions are selected from thegroup consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52,L53, L55, L91, L92, L93, L94 and L96 and the other characteristic isselected from 1) preservation of non-crossreactivity with other proteinsor human tissues, 2) preservation of epitope recognition, i.e.recognizing p40 epitope preferably in the context of the p70 p40/p35heterodimer preventing binding interference from free, soluble p40and/or 3) to produce an antibody with a close to germline immunoglobulinsequence. In another preferred embodiment, the hypermutation positionsare selected from the group consisting of H30, H31, H31B, H32, H52, H56,H58, L30, L31, L32, L53 and L93 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence. Ina more preferred embodiment the residues for selective mutagenesis areselected from the preferred selective mutagenesis positions from thegroup consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,L32, L50, L91, L92, L93, L94 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence. Ina more preferred embodiment, the contact positions are selected from thegroup consisting of L50 and L94 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence.

In another embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a recombinant parent antibody or antigen-binding portionthereof; that was obtained by selection in a phage-display system butwhose activity cannot be further improved by mutagenesis in saidphage-display system;

b) selecting a preferred selective mutagenesis position, contact orhypermutation position within a complementarity determining region (CDR)for mutation, thereby identifying a selected preferred selectivemutagenesis position contact or hypermutation position;

c) individually mutating said selected preferred selective mutagenesisposition, contact or hypermutation position to at least two other aminoacid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof, and expressing said panel in anon-phage display system;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof for at least one other property or characteristic,wherein the property or characteristic is one that needs to be retained,until an antibody, or antigen-binding portion thereof, with an improvedactivity and at least one retained property or characteristic, relativeto the parent antibody, or antigen-binding portion thereof, is obtained.

f) repeating steps a) through e) for at least one other preferredselective mutagenesis position, contact or hypermutation position;

g) combining, in the parent antibody, or antigen-binding portionthereof, at least two individual activity enhancing amino acid residuesshown to have improved activity and at least on retained othercharacteristic, to form combination antibodies, or antigen-bindingportions thereof; and

h) evaluating the activity of the combination antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof; until an antibody, or antigen-bindingportion thereof, with an improved activity and at least one retainedproperty or characteristic, relative to the parent antibody, orantigen-binding portion thereof, is obtained.

In a preferred embodiment, the contact positions are selected from thegroup consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52,L53, L55, L91, L92, L93, L94 and L96 and the other characteristic isselected from 1) preservation of non-crossreactivity with other proteinsor human tissues, 2) preservation of epitope recognition, i.e.recognizing p40 epitope preferably in the context of the p70 p40/p35heterodimer preventing binding interference from free, soluble p40and/or 3) to produce an antibody with a close to germline immunoglobulinsequence. In another preferred embodiment, the hypermutation positionsare selected from the group consisting of H30, H31, H31B, H32, H52, H56,H58, L30, L31, L32, L53 and L93 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence. Ina more preferred embodiment the residues for selective mutagenesis areselected from the preferred selective mutagenesis positions from thegroup consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,L32, L50, L91, L92, L93, L94 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence. Ina more preferred embodiment, the contact positions are selected from thegroup consisting of L50 and L94 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence.

In another embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50,H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

c) individually mutating said selected position to at least two otheramino acid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof, for changes in at least one other property orcharacteristic;

until an antibody, or antigen-binding portion thereof, with an improvedactivity, relative to the parent antibody, or antigen-binding portionthereof, is obtained.

Preferably, the other characteristic or property is selected from 1)preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence

In another embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50,H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

c) individually mutating said selected position to at least two otheramino acid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof, thereby identifying an activityenhancing amino acid residue;

e) repeating steps b) through d) for at least one other CDR positionwhich is neither the position selected under b) nor a position at H30,H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96,H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,L94 and L96;

f) combining, in the parent antibody, or antigen-binding portionthereof, at least two individual activity enhancing amino acid residuesshown to have improved activity, to form combination antibodies, orantigen-binding portions thereof; and

g) evaluating the activity of the combination antibodies, orantigen-binding portions thereof with two activity enhancing amino acidresidues, relative to the parent antibody or antigen-binding portionthereof until an antibody, or antigen-binding portion thereof, with animproved activity, relative to the parent antibody, or antigen-bindingportion thereof, is obtained.

In another embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a recombinant parent antibody or antigen-binding portionthereof; that was obtained by selection in a phage-display system butwhose activity cannot be further improved by mutagenesis in saidphage-display system;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50,H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,L34, L50, L52, L53, L55, L91, L92, L93, L94 and;

c) individually mutating said selected contact or hypermutation positionto at least two other amino acid residues to thereby create a panel ofmutated antibodies, or antigen-binding portions thereof, and expressingsaid panel in a non-phage display system;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof, for changes in at least one other property orcharacteristic until an antibody, or antigen-binding portion thereof,with an improved activity, relative to the parent antibody, orantigen-binding portion thereof, is obtained.

Preferably, the other characteristic or property is selected from 1)preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence.

In another embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof thatwas obtained by selection in a phage-display system but whose activitycannot be further improved by mutagenesis in said phage-display system;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50,H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

c) individually mutating said selected position to at least two otheramino acid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof and expression in a non-phage displaysystem;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) repeating steps b) through d) for at least one other position withinthe CDR which is neither the position selected under b) nor a positionat H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58,H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91,L92, L93, L94;

f) combining, in the parent antibody, or antigen-binding portionthereof, at least two individual activity enhancing amino acid residuesshown to have improved activity, to form combination antibodies, orantigen-binding portions thereof; and

g) evaluating the activity and other property or characteristic of thecombination antibodies, or antigen-binding portions thereof with twoactivity enhancing amino acid residues, relative to the parent antibodyor antigen-binding portion thereof; until an antibody, orantigen-binding portion thereof, with an improved activity, relative tothe parent antibody, or antigen-binding portion thereof, is obtained.

Preferably, the other characteristic or property is selected from 1)preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence.

In another embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50,H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

c) individually mutating said selected position to at least two otheramino acid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof, thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies or antigen-bindingportions thereof, relative to the parent antibody or antigen-portionthereof, for changes in at least one other property or characteristic;

f) repeating steps b) through e) for at least one other CDR positionwhich is neither the position selected under b) nor a position at H30,H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96,H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,L94 and L96;

g) combining, in the parent antibody, or antigen-binding portionthereof, at least two individual activity enhancing amino acid residuesshown to have improved activity and not affecting at least one otherproperty or characteristic, to form combination antibodies, orantigen-binding portions thereof; and

h) evaluating the activity and the retention of at least one othercharacteristic or property of the combination antibodies, orantigen-binding portions thereof with two activity enhancing amino acidresidues, relative to the parent antibody or antigen-binding portionthereof until an antibody, or antigen-binding portion thereof, with animproved activity and at least one retained property or characteristic,relative to the parent antibody, or antigen-binding portion thereof, isobtained.

In another embodiment the invention provides a method to improve theaffinity of an antibody or antigen-binding portion thereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof thatwas obtained by selection in a phage-display system but whose activitycannot be further improved by mutagenesis in said phage-display system;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50,H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

c) individually mutating said selected position to at least two otheramino acid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof and expression in a non-phage displaysystem;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof, for changes in at least one other characteristic orproperty until an antibody, or antigen-binding portion thereof, with animproved activity, relative to the parent antibody, or antigen-bindingportion thereof, is obtained.

In another embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation at a position other than H30, H31, H31B, H32,H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101,L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

c) individually mutating said selected position to at least two otheramino acid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof, thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies or antigen-bindingportions thereof, relative to the parent antibody or antigen-portionthereof, for changes in at least one other property or characteristic;

f) repeating steps b) through e) for at least one other CDR positionwhich is neither the position selected under b) nor a position at H30,H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96,H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,L94 and L96;

g) combining, in the parent antibody, or antigen-binding portionthereof, at least two individual activity enhancing amino acid residuesshown to have improved activity but not affecting at least one otherproperty or characteristic, to form combination antibodies, orantigen-binding portions thereof with at least one retained property orcharacteristic; and

h) evaluating the activity and the retention of at least one property ofcharacteristic of the combination antibodies, or antigen-bindingportions thereof with two activity enhancing amino acid residues,relative to the parent antibody or antigen-binding portion thereof untilan antibody, or antigen-binding portion thereof, with an improvedactivity and at least one retained property or characteristic, relativeto the parent antibody, or antigen-binding portion thereof, is obtained.

Preferably, the other characteristic or property is selected from 1)preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence

In another embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, withoutaffecting other characteristics, comprising:

a) providing a parent antibody or antigen-binding portion thereof;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50,H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

c) individually mutating said selected position to at least two otheramino acid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof, for changes in at least one other property orcharacteristic until an antibody, or antigen-binding portion thereof,with an improved activity and retained other characteristic or property,relative to the parent antibody, or antigen-binding portion thereof, isobtained.

In another embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof thatwas obtained by selection in a phage-display system but whose activitycannot be further improved by mutagenesis in said phage-display system;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50,H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

c) individually mutating said selected position to at least two otheramino acid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof and expression in a non-phage displaysystem;

d) evaluating the activity and retention of at least one othercharacteristic or property of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof, thereby identifying an activityenhancing amino acid residue;

e) repeating steps b) through d) for at least one other CDR positionwhich is neither the position selected under b nor other than H30, H31,H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94and L96;

f) combining, in the parent antibody, or antigen-binding portionthereof, at least two individual activity enhancing amino acid residuesshown to have improved activity and not to affect at least one othercharacteristic or property, to form combination antibodies, orantigen-binding portions thereof; and

g) evaluating the activity and retention of at least one othercharacteristic or property of the combination antibodies, orantigen-binding portions thereof with two activity enhancing amino acidresidues, relative to the parent antibody or antigen-binding portionthereof until an antibody, or antigen-binding portion thereof, with animproved activity and at least one other retained characteristic orproperty, relative to the parent antibody, or antigen-binding portionthereof, is obtained.

Preferably, the other characteristic or property is selected from 1)preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show the heavy chain variable region amino acid sequencealignments of a series of human antibodies that bind human IL-12compared to germline sequences Cos-3/JH3 and Dp118 Lv1042. Kabatnumbering is used to identify amino acid positions. For the Joe 9 wildtype, the full sequence is shown. For the other antibodies, only thoseamino acids positions that differ from Joe 9 wild type are shown.

FIGS. 1C-1D show the light chain variable region amino acid sequencealignments of a series of human antibodies that bind human IL-12. Kabatnumbering is used to identify amino acid positions. For the Joe 9 wildtype, the full sequence is shown. For the other antibodies, only thoseamino acids positions that differ from Joe 9 wild type are shown.

FIGS. 2A-2E show the CDR positions in the heavy chain of the Y61antibody that were mutated by site-directed mutagenesis and therespective amino acid substitutions at each position. The graphs at theright of the figures show the off-rates for the substituted antibodies(black bars) as compared to unmutated Y61 (open bar).

FIGS. 2F-2H show the CDR positions in the light chain of the Y61antibody that were mutated by site-directed mutagenesis and therespective amino acid substitutions at each position. The graphs at theright of the figures show the off-rates for the substituted antibodies(black bars) as compared to unmutated Y61 (open bar).

FIGS. 3A-3B demonstrate the in vivo efficacy of the human anti-IL-12antibody J695, on plasma neopterin levels in cynomolgus monkeys.

FIG. 4 shows a graph of mean arthritic score versus days afterimmunization of mice with collagen, demonstrating that treatment withC17.15 significantly decreases arthritis-related symptoms as compared totreatment with rat IgG.

DETAILED DESCRIPTION OF THE INVENTION

In order that the present invention may be more readily understood,certain terms are first defined.

The term “activity enhancing amino acid residue” includes an amino acidresidue which improves the activity of the antibody. It should beunderstood that the activity enhancing amino acid residue may replace anamino acid residue at a contact, hypermutation or preferred selectivemutagenesis position and, further, more than one activity enhancingamino acid residue can be present within one or more CDRs. An activityenchancing amino acid residue include, an amino acid residue thatimproves the binding specificity/affinity of an antibody, for exampleanti-human IL-12 antibody binding to human IL-12. The activity enhancingamino acid residue is also intended to include an amino acid residuethat improves the neutralization potency of an antibody, for example,the human IL-12 antibody which inhibits human IL-12.

The term “antibody” includes an immunoglobulin molecule comprised offour polypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as HCVR or VH) and aheavy chain constant region. The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as LCVRor VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDRs), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The term “antigen-binding portion” of an antibody (or “antibodyportion”) includes fragments of an antibody that retain the ability tospecifically bind to an antigen (e.g., hIL-12). It has been shown thatthe antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodiesare also intended to be encompassed within the term “antigen-bindingportion” of an antibody. Other forms of single chain antibodies, such asdiabodies are also encompassed. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc.Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994)Structure 2:1121-1123). Still further, an antibody or antigen-bindingportion thereof may be part of a larger immunoadhesion molecules, formedby covalent or non-covalent association of the antibody or antibodyportion with one or more other proteins or peptides. Examples of suchimmunoadhesion molecules include use of the streptavidin core region tomake a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein. Preferred antigen binding portions are completedomains or pairs of complete domains.

The term “backmutation” refers to a process in which some or all of thesomatically mutated amino acids of a human antibody are replaced withthe corresponding germline residues from a homologous germline antibodysequence. The heavy and light chain sequences of the human antibody ofthe invention are aligned separately with the germline sequences in theVBASE database to identify the sequences with the highest homology.Differences in the human antibody of the invention are returned to thegermline sequence by mutating defined nucleotide positions encoding suchdifferent amino acid. The role of each amino acid thus identified ascandidate for backmutation should be investigated for a direct orindirect role in antigen binding and any amino acid found after mutationto affect any desirable characteristic of the human antibody should notbe included in the final human antibody; as an example, activityenhancing amino acids identified by the selective mutagenesis approachwill not be subject to backmutation. To minimize the number of aminoacids subject to backmutation those amino acid positions found to bedifferent from the closest germline sequence but identical to thecorresponding amino acid in a second germline sequence can remain,provided that the second germline sequence is identical and colinear tothe sequence of the human antibody of the invention for at least 10,preferably 12 amino acids, on both sides of the amino acid in question.Backmuation may occur at any stage of antibody optimization; preferably,backmutation occurs directly before or after the selective mutagenesisapproach. More preferably, backmutation occurs directly before theselective mutagenesis approach.

The phrase “human interleukin 12” (abbreviated herein as hIL-12, orIL-12), as used herein, includes a human cytokine that is secretedprimarily by macrophages and dendritic cells. The term includes aheterodimeric protein comprising a 35 kD subunit (p35) and a 40 kDsubunit (p40) which are both linked together with a disulfide bridge.The heterodimeric protein is referred to as a “p70 subunit”. Thestructure of human IL-12 is described further in, for example,Kobayashi, et al. (1989) J. Exp Med. 170:827-845; Seder, et al. (1993)Proc. Natl. Acad. Sci. 90:10188-10192; Ling, et al. (1995) J. Exp Med.154:116-127; Podlaski, et al. (1992) Arch. Biochem. Biophys.294:230-237. The term human IL-12 is intended to include recombinanthuman IL-12 (rh IL-12), which can be prepared by standard recombinantexpression methods.

The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

The Kabat numbering is used herein to indicate the positions of aminoacid modifications made in antibodies of the invention. For example, theY61 anti-IL-12 antibody can be mutated from serine (S) to glutamic acid(E) at position 31 of the heavy chain CDR1 (H31S→E), or glycine (G) canbe mutated to tyrosine (Y) at position 94 of the light chain CDR3(L94G→Y).

The term “human antibody” includes antibodies having variable andconstant regions corresponding to human germline immunoglobulinsequences as described by Kabat et al. (See Kabat, et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3. Themutations preferably are introduced using the “selective mutagenesisapproach” described herein. The human antibody can have at least oneposition replaced with an amino acid residue, e.g., an activityenhancing amino acid residue which is not encoded by the human germlineimmunoglobulin sequence. The human antibody can have up to twentypositions replaced with amino acid residues which are not part of thehuman germline immunoglobulin sequence. In other embodiments, up to ten,up to five, up to three or up to two positions are replaced. In apreferred embodiment, these replacements are within the CDR regions asdescribed in detail below. However, the term “human antibody”, as usedherein, is not intended to include antibodies in which CDR sequencesderived from the germline of another mammalian species, such as a mouse,have been grafted onto human framework sequences.

The phrase “recombinant human antibody” includes human antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell (described further in Section II, below),antibodies isolated from a recombinant, combinatorial human antibodylibrary (described further in Section III, below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor, L. D., et al. (1992) Nucl. AcidsRes. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences (See Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).In certain embodiments, however, such recombinant human antibodies aresubjected to in vitro mutagenesis (or, when an animal transgenic forhuman Ig sequences is used, in vivo somatic mutagenesis) and thus theamino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangermline VH and VL sequences, may not naturally exist within the humanantibody germline repertoire in vivo. In certain embodiments, however,such recombinant antibodies are the result of selective mutagenesisapproach or backmutation or both.

An “isolated antibody” includes an antibody that is substantially freeof other antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds hIL-12 is substantially freeof antibodies that specifically bind antigens other than hIL-12). Anisolated antibody that specifically binds hIL-12 may bind IL-12molecules from other species (discussed in further detail below).Moreover, an isolated antibody may be substantially free of othercellular material and/or chemicals.

A “neutralizing antibody” (or an “antibody that neutralized hIL-12activity”) includes an antibody whose binding to hIL-12 results ininhibition of the biological activity of hIL-12. This inhibition of thebiological activity of hIL-12 can be assessed by measuring one or moreindicators of hIL-12 biological activity, such as inhibition of humanphytohemagglutinin blast proliferation in a phytohemagglutinin blastproliferation assay (PHA), or inhibition of receptor binding in a humanIL-12 receptor binding assay (see Example 3-Interferon-gamma InductionAssay). These indicators of hIL-12 biological activity can be assessedby one or more of several standard in vitro or in vivo assays known inthe art (see Example 3).

The term “activity” includes activities such as the bindingspecificity/affinity of an antibody for an antigen, for example, ananti-hIL-12 antibody that binds to an IL-12 antigen and/or theneutralizing potency of an antibody, for example, an anti-hIL-12antibody whose binding to hIL-12 inhibits the biological activity ofhIL-12, e.g. inhibition of PHA blast proliferation or inhibition ofreceptor binding in a human IL-12 receptor binding assay (see Example3).

The phrase “surface plasmon resonance” includes an optical phenomenonthat allows for the analysis of real-time biospecific interactions bydetection of alterations in protein concentrations within a biosensormatrix, for example using the BIAcore system (Pharmacia Biosensor AB,Uppsala, Sweden and Piscataway, N.J.). For further descriptions, seeExample 5 and Jönsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26;Jönsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., etal. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991)Anal. Biochem. 198:268-277.

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

The phrase “nucleic acid molecule” includes DNA molecules and RNAmolecules. A nucleic acid molecule may be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

The phrase “isolated nucleic acid molecule”, as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., VH, VL,CDR3) that bind hIL-12 including “isolated antibodies”), includes anucleic acid molecule in which the nucleotide sequences encoding theantibody or antibody portion are free of other nucleotide sequencesencoding antibodies or antibody portions that bind antigens other thanhIL-12, which other sequences may naturally flank the nucleic acid inhuman genomic DNA. Thus, for example, an isolated nucleic acid of theinvention encoding a VH region of an anti-IL-12 antibody contains noother sequences encoding other VH regions that bind antigens other thanIL-12. The phrase “isolated nucleic acid molecule” is also intended toinclude sequences encoding bivalent, bispecific antibodies, such asdiabodies in which VH and VL regions contain no other sequences otherthan the sequences of the diabody.

The term “vector” includes a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “expressionvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The phrase “recombinant host cell” (or simply “host cell”) includes acell into which a recombinant expression vector has been introduced. Itshould be understood that such terms are intended to refer not only tothe particular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

The term “modifying”, as used herein, is intended to refer to changingone or more amino acids in the antibodies or antigen-binding portionsthereof. The change can be produced by adding, substituting or deletingan amino acid at one or more positions. The change can be produced usingknown techniques, such as PCR mutagenesis.

The phrase “contact position” includes an amino acid position of in theCDR1, CDR2 or CDR3 of the heavy chain variable region or the light chainvariable region of an antibody which is occupied by an amino acid thatcontacts antigen in one of the twenty-six known antibody-antigenstructures. If a CDR amino acid in any of the 26 known solved structuresof antibody-antigen complexes contacts the antigen, then that amino acidcan be considered to occupy a contact position. Contact positions have ahigher probability of being occupied by an amino acid which contactantigen than non-contact positions. Preferably a contact position is aCDR position which contains an amino acid that contacts antigen ingreater than 3 of the 26 structures (>11.5%). Most preferably a contactposition is a CDR position which contains an amino acid that contactsantigen in greater than 8 of the 25 structures (>32%).

The term “hypermutation position” includes an amino acid residue thatoccupies position in the CDR1, CDR2 or CDR3 region of the heavy chainvariable region or the light chain variable region of an antibody thatis considered to have a high frequency or probability for somatichypermutation during in vivo affinity maturation of the antibody. “Highfrequency or probability for somatic hypermutation” includes frequenciesor probabilities of a 5 to about 40% chance that the residue willundergo somatic hypermutation during in vivo affinity maturation of theantibody. It should be understood that all ranges within this statedrange are also intended to be part of this invention, e.g., 5 to about30%, e.g., 5 to about 15%, e.g., 15 to about 30%.

The term “preferred selective mutagenesis position” includes an aminoacid residue that occupies a position in the CDR1, CDR2 or CDR3 regionof the heavy chain variable region or the light chain variable regionwhich can be considered to be both a contact and a hypermutationposition.

The phrase “selective mutagenesis approach” includes a method ofimproving the activity of an antibody by selecting and individuallymutating CDR amino acids at at least one preferred selective mutagenesisposition, hypermutation, and/or contact position. A “selectivelymutated” human antibody is an antibody which contains a mutation at aposition selected using a selective mutagenesis approach. In anotherembodiment, the selective mutagenesis approach is intended to provide amethod of preferentially mutating selected individual amino acidresidues in the CDR1, CDR2 or CDR3 of the heavy chain variable region(hereinafter H1, H2, and H3, respectively), or the CDR1, CDR2 or CDR3 ofthe light chain variable region (hereinafter referred to as L1, L2, andL3, respectively) of an antibody. Amino acid residues may be selectedfrom preferred selective mutagenesis positions, contact positions, orhypermutation positions. Individual amino acids are selected based ontheir position in the light or heavy chain variable region. It should beunderstood that a hypermutation position can also be a contact position.In an embodiment, the selective mutagenesis approach is a “targetedapproach”. The language “targeted approach” is intended to include amethod of preferentially mutating selected individual amino acidresidues in the CDR1, CDR2 or CDR3 of the heavy chain variable region orthe CDR1, CDR2 or CDR3 of the light chain variable region of an antibodyin a targeted manner, e.g., a “Group-wise targeted approach” or“CDR-wise targeted approach”. In the “Group-wise targeted approach”,individual amino acid residues in particular groups are targeted forselective mutations including groups I (including L3 and H3), II(including H2 and L1) and III (including L2 and H1), the groups beinglisted in order of preference for targeting. In the “CDR-wise targetedapproach”, individual amino acid residues in particular CDRs aretargeted for selective mutations with the order of preference fortargeting as follows: H3, L3, H2, L1, H1 and L2. The selected amino acidresidue is mutated, e.g., to at least two other amino acid residues, andthe effect of the mutation on the activity of the antibody isdetermined. Activity is measured as a change in the bindingspecificity/affinity of the antibody, and/or neutralization potency ofthe antibody. It should be understood that the selective mutagenesisapproach can be used for the optimization of any antibody derived fromany source including phage display, transgenic animals with human IgGgermline genes, human antibodies isolated from human B-cells.Preferably, the selective mutagenesis approach is used on antibodieswhich can not be optimized further using phage display technology. Itshould be understood that antibodies from any source including phagedisplay, transgenic animals with human IgG germline genes, humanantibodies isolated from human B-cells can be subject to backmutationprior to or after the selective mutagenesis approach.

The term “activity enhancing amino acid residue” includes an amino acidresidue which improves the activity of the antibody. It should beunderstood that the activity enhancing amino acid residue may replace anamino acid residue at a preferred selective mutagenesis position,contact position, or a hypermutation position and, further, more thanone activity enhancing amino acid residue can be present within one ormore CDRs. An activity enchancing amino acid residue include, an aminoacid residue that improves the binding specificity/affinity of anantibody, for example anti-human IL-12 antibody binding to human IL-12.The activity enhancing amino acid residue is also intended to include anamino acid residue that improves the neutralization potency of anantibody, for example, the human IL-12 antibody which inhibits humanIL-12.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Human Antibodies that Bind Human IL-12

This invention provides isolated human antibodies, or antigen-bindingportions thereof, that bind to human IL-12. Preferably, the humanantibodies of the invention are recombinant, neutralizing humananti-hIL-12 antibodies. Antibodies of the invention that bind to humanIL-12 can be selected, for example, by screening one or more human V_(L)and V_(H) cDNA libraries with hIL-12, such as by phage displaytechniques as described in Example 1. Screening of human V_(L) and V_(H)cDNA libraries initially identified a series of anti-IL-12 antibodies ofwhich one antibody, referred to herein as “Joe 9” (or “Joe 9 wildtype”), was selected for further development. Joe 9 is a relatively lowaffinity human IL-12 antibody (e.g., a K_(off) of about 0.1 sec⁻¹), yetis useful for specifically binding and detecting hIL-12. The affinity ofthe Joe 9 antibody was improved by conducting mutagenesis of the heavyand light chain CDRs, producing a panel of light and heavy chainvariable regions that were “mixed and matched” and further mutated,leading to numerous additional anti-hIL-12 antibodies with increasedaffinity for hIL-12 (see Example 1, Table 2 (see Appendix A) and thesequence alignments of FIGS. 1A-D).

Of these antibodies, the human anti-hIL-12 antibody referred to hereinas Y61 demonstrated a significant improvement in binding affinity (e.g.,a K_(off) of about 2×10⁻⁴ sec⁻¹). The Y61 anti-hIL-12 antibody wasselected for further affinity maturation by individually mutatingspecific amino acids residues within the heavy and light chain CDRsAmino acids residues of Y61 were selected for site-specific mutation(selective mutagenesis approach) based on the amino acid residueoccupying a preferred selective mutagenesis position, contact and/or ahypermutation position. A summary of the substitutions at selectedpositions in the heavy and light chain CDRs is shown in FIGS. 2A-2H. Apreferred recombinant neutralizing antibody of the invention, referredto herein as J695, resulted from a Gly to Tyr substitution at position50 of the light chain CDR2 of Y61, and a Gly to Tyr substitution atposition 94 of the light chain CDR3 of Y61.

Amino acid sequence alignments of the heavy and light chain variableregions of a panel of anti-IL-12 antibodies of the invention, on thelineage from Joe 9 wild type to J695, are shown in FIGS. 1A-1D. Thesesequence alignments allowed for the identification of consensussequences for preferred heavy and light chain variable regions ofantibodies of the invention that bind hIL-12, as well as consensussequences for the CDR3, CDR2, and CDR1, on the lineage from Joe 9 toJ695. Moreover, the Y61 mutagenesis analysis summarized in FIGS. 2A-2Hallowed for the identification of consensus sequences for heavy andlight chain variable regions that bind hIL-12, as well as consensussequences for the CDR3, CDR2, and CDR1 that bind hIL-12 on the lineagefrom Y61 to J695 that encompasses sequences with modifications from Y61yet that retain good hIL-12 binding characteristics. Preferred CDR, VHand VL sequences of the invention (including consensus sequences) asidentified by sequence identifiers in the attached Sequence Listing, aresummarized below.

SEQ ID ANTIBODY NO: CHAIN REGION SEQUENCE  1 Consensus CDR H3(H/S)-G-S-(H/Y)-D-(N/T/Y) Joe 9 to J695  2 Consensus CDR L3Q-(S/T)-Y-(D/E)-(S/R/K)-(S/G/Y)- Joe 9 to J695(L/F/T/S)-(R/S/T/W/H)-(G/P)- (S/T/A/L)-(R/S/M/T/L)-(V/I/T/M/L)  3Consensus CDR H2 F-I-R-Y-D-G-S-N-K-Y-Y-A-D-S-V-K-G Joe 9 to J695  4Consensus CDR L2 (G/Y)-N-(D/S)-(Q/N)-R-P-S Joe 9 to J695  5 ConsensusCDR H1 F-T-F-S-(S/E)-Y-G-M-H Joe 9 to J695  6 Consensus CDR L1(S/T)-G-(G/S)-(R/S)-S-N-I-(G/V)- Joe 9 to J695(S/A)-(N/G/Y)-(T/D)-V-(K/H)  7 Consensus VH (full VH sequence; seeJoe 9 to J695 sequence listing)  8 Consensus VL (full VL sequence; seeJoe 9 to J695 sequence listing)  9 Consensus CDR H3H-(G/V/C/H)-(S/T)-(H/T/V/R/I)- Y61 to J695 (D/S)-(N/K/A/T/S/F/W/H) 10Consensus CDR L3 Q-S-Y-(D/S)-(Xaa)- Y61 to J695(G/D/Q/L/F/R/H/N/Y)-T-H-P-A-L-L 11 Consensus CDR H2(F/T/Y)-I-(R/A)-Y-(D/S/E/A)-(G/R)- Y61 to J695S-(Xaa)-K-(Y/E)-Y-A-D-S-V-K-G 12 Consensus CDR L2(G/Y/S/T/N/Q)-N-D-Q-R-P-S Y61 to J695 13 Consensus CDR H1F-T-F-(Xaa)-(Xaa)-(Y/H)- Y61 to J695 (G/M/A/N/S)-M-H 14 Consensus CDR L1S-G-G-R-S-N-I-G-(S/C/R/N/D/T)- Y61 to J695 (N/M/I)-(T/Y/D/H/K/P)-V-K 15Consensus VH (full VH sequence; see Y61 to J695 sequence listing) 16Consensus VL (full VL sequence; see Y61 to J695 sequence listing) 17 Y61CDR H3 H-G-S-H-D-N 18 Y61 CDR L3 Q-S-Y-D-R-G-T-H-P-A-L-L 19 Y61 CDR H2F-I-R-Y-D-G-S-N-K-Y-Y-A-D-S-V-K-G 20 Y61 CDR L2 G-N-D-Q-R-P-S 21 Y61CDR H1 F-T-F-S-S-Y-G-M-H 22 Y61 CDR L1 S-G-G-R-S-N-I-G-S-N-T-V-K 23 Y61VH (full VH sequence; see sequence listing) 24 Y61 VL(full VL sequence; see sequence listing) 25 J695 CDR H3 H-G-S-H-D-N 26J695 CDR L3 Q-S-Y-D-R-Y-T-H-P-A-L-L 27 J695 CDR H2F-I-R-Y-D-G-S-N-K-Y-Y-A-D-S-V-K-G 28 J695 CDR L2 Y-N-D-Q-R-P-S 29 J695CDR H1 F-T-F-S-S-Y-G-M-H 30 J695 CDR L1 S-G-S-R-S-N-I-G-S-N-T-V-K 31J695 VH (full VH sequence; see sequence listing) 32 J695 VL(full VL sequence; see sequence listing)

Antibodies produced from affinity maturation of Joe 9 wild type werefunctionally characterized by surface plasmon resonance analysis todetermine the K_(d) and K_(off) rate. A series of antibodies wereproduced having a K_(off) rate within the range of about 0.1 s⁻¹ toabout 1×10⁻⁵ s⁻¹, and more preferably a K_(off) of about 1×10⁻⁴ s⁻¹ to1×10⁻⁵ s⁻¹ or less. Antibodies were also characterized in vitro fortheir ability to inhibit phytohemagglutinin (PHA) blast proliferation,as described in Example 3. A series of antibodies were produced havingan IC₅₀ value in the range of about 1×10⁻⁶ M to about 1×10⁻¹¹ M, morepreferably about 1×10⁻¹⁰ M to 1×10⁻¹¹ M or less.

Accordingly, in one aspect, the invention provides an isolated humanantibody, or antigen-binding portion thereof, that binds to human IL-12and dissociates from human IL-12 with a K_(off) rate constant of 0.1 s⁻¹or less, as determined by surface plasmon resonance, or which inhibitsphytohemagglutinin blast proliferation in an in vitro phytohemagglutininblast proliferation assay (PHA assay) with an IC₅₀ of 1×10⁻⁶ M or less.In preferred embodiments, the isolated human IL-12 antibody, or anantigen-binding portion thereof, dissociates from human IL-12 with aK_(off) rate constant of 1×10⁻² s⁻¹ or less, or inhibitsphytohemagglutinin blast proliferation in an in vitro PHA assay with anIC₅₀ of 1×10⁻⁷ M or less. In more preferred embodiments, the isolatedhuman IL-12 antibody, or an antigen-binding portion thereof, dissociatesfrom human IL-12 with a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, orinhibits phytohemagglutinin blast proliferation in an in vitro PHA assaywith an IC₅₀ of 1×10⁻⁸ M or less. In more preferred embodiments, theisolated human IL-12 antibody, or an antigen-binding portion thereof,dissociates from human IL-12 with a K_(off) rate constant of 1×10⁻⁴ s⁻¹or less, or inhibits phytohemagglutinin blast proliferation in an invitro PHA assay with an IC₅₀ of 1×10⁻⁹ M or less. In more preferredembodiments, the isolated human IL-12 antibody, or an antigen-bindingportion thereof, dissociates from human IL-12 with a K_(off) rateconstant of 1×10⁻⁵ s⁻¹ or less, or inhibits phytohemagglutinin blastproliferation in an in vitro PHA assay with an IC₅₀ of 1×10⁻¹⁰ M orless. In even more preferred embodiments, the isolated human IL-12antibody, or an antigen-binding portion thereof, dissociates from humanIL-12 with a K_(off) rate constant of 1×10⁻⁵ s⁻¹ or less, or inhibitsphytohemagglutinin blast proliferation in an in vitro PHA assay with anIC₅₀ of 1×10⁻¹¹ M or less.

The dissociation rate constant (K_(off)) of an IL-12 antibody can bedetermined by surface plasmon resonance (see Example 5). Generally,surface plasmon resonance analysis measures real-time bindinginteractions between ligand (recombinant human IL-12 immobilized on abiosensor matrix) and analyte (antibodies in solution) by surfaceplasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor,Piscataway, N.J.). Surface plasmon analysis can also be performed byimmobilizing the analyte (antibodies on a biosensor matrix) andpresenting the ligand (recombinant IL-12 in solution). Neutralizationactivity of IL-12 antibodies, or antigen binding portions thereof, canbe assessed using one or more of several suitable in vitro assays (seeExample 3).

It is well known in the art that antibody heavy and light chain CDRsplay an important role in the binding specificity/affinity of anantibody for an antigen. Accordingly, the invention encompasses humanantibodies having light and heavy chain CDRs of Joe 9, as well as otherantibodies having CDRs that have been modified to improve the bindingspecificity/affinity of the antibody. As demonstrated in Example 1, aseries of modifications to the light and heavy chain CDRs results inaffinity maturation of human anti-hIL-12 antibodies. The heavy and lightchain variable region amino acid sequence alignments of a series ofhuman antibodies ranging from Joe 9 wild type to J695 that bind humanIL-12 is shown in FIGS. 1A-1D. Consensus sequence motifs for the CDRs ofantibodies can be determined from the sequence alignment (as summarizedin the table above). For example, a consensus motif for the VH CDR3 ofthe lineage from Joe 9 to J695 comprises the amino acid sequence:(H/S)-G-S-(H/Y)-D-(N/T/Y) (SEQ ID NO: 1), which encompasses amino acidsfrom position 95 to 102 of the consensus HCVR shown in SEQ ID NO: 7. Aconsensus motif for the VL CDR3 comprises the amino acid sequence:Q-(S/T)-Y-(D/E)-(S/R/K)-(S/G/Y)-(L/F/T/S)-(R/S/T/W/H)-(G/P)-(S/T/A/L)-(R/S/M/T/L-V/I/T/M/L)(SEQ ID NO: 2), which encompasses amino acids from position 89 to 97 ofthe consensus LCVR shown in SEQ ID NO: 8.

Accordingly, in another aspect, the invention provides an isolated humanantibody, or an antigen-binding portion thereof, which has the followingcharacteristics:

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁶ M or less;

b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ IDNO: 1; and

c) has a light chain CDR3 comprising the amino acid sequence of SEQ IDNO: 2.

In a preferred embodiment, the antibody further comprises a VH CDR2comprising the amino acid sequence: F-I-R-Y-D-G-S-N-K-Y-Y-A-D-S-V-K-G(SEQ ID NO: 3) (which encompasses amino acids from position 50 to 65 ofthe consensus HCVR comprising the amino acid sequence SEQ ID NO: 7) andfurther comprises a VL CDR2 comprising the amino acid sequence:(G/Y)-N-(D/S)-(Q/N)-R-P-S(SEQ ID NO: 4) (which encompasses amino acidsfrom position 50 to 56 of the consensus LCVR comprising the amino acidsequence SEQ ID NO: 8).

In another preferred embodiment, the antibody further comprises a VHCDR1 comprising the amino acid sequence: F-T-F-S-(S/E)-Y-G-M-H (SEQ IDNO: 5) (which encompasses amino acids from position 27 to 35 of theconsensus HCVR comprising the amino acid sequence SEQ ID NO: 7) andfurther comprises a VL CDR1 comprising the amino acid sequence:(S/T)-G-(G/S)-(R/S)-S-N-I-(G/V)-(S/A)-(N/G/Y)-(T/D)-V-(K/H) (SEQ ID NO:6) (which encompasses amino acids from position 24 to 34 of theconsensus LCVR comprising the amino acid sequence SEQ ID NO: 8).

In yet another preferred embodiment, the antibody of the inventioncomprises a HCVR comprising the amino acid sequence of SEQ ID NO: 7 anda LCVR comprising the amino acid sequence of SEQ ID NO: 8.

Additional consensus motifs can be determined based on the mutationalanalysis performed on Y61 that led to the J695 antibody (summarized inFIGS. 2A-2H). As demonstrated by the graphs shown in FIGS. 2A-2H,certain residues of the heavy and light chain CDRs of Y61 were amenableto substitution without significantly impairing the hIL-12 bindingproperties of the antibody. For example, individual substitutions atposition 30 in CDR H1 with twelve different amino acid residues did notsignificantly reduce the K_(off) rate of the antibody, indicating thatis position is amenable to substitution with a variety of differentamino acid residues. Thus, based on the mutational analysis (i.e.,positions within Y61 that were amenable to substitution by other aminoacid residues) consensus motifs were determined. The consensus motifsfor the heavy and light chain CDR3s are shown in SEQ ID NOs: 9 and 10,respectively, consensus motifs for the heavy and light chain CDR2s areshown in SEQ ID NOs: 11 and 12, respectively, and consensus motifs forthe heavy and light chain CDR1s are shown in SEQ ID NOs: 13 and 14,respectively. Consensus motifs for the VH and VL regions are shown inSEQ ID NOs: 15 and 16, respectively.

Accordingly, in one aspect, the invention features an isolated humanantibody, or an antigen-binding portion thereof, which has the followingcharacteristics:

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁹ M or less;

b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ IDNO: 9; and

c) has a light chain CDR3 comprising the amino acid sequence of SEQ IDNO: 10.

In a preferred embodiment, the antibody further comprises a VH CDR2comprising the amino acid sequence of SEQ ID NO: 11 and furthercomprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 12.

In another preferred embodiment, the antibody further comprises a VHCDR1 comprising the amino acid sequence of SEQ ID NO: 13 and furthercomprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 14.

In yet another preferred embodiment, the antibody of the inventioncomprises a HCVR comprising the amino acid sequence of SEQ ID NO: 15 anda LCVR comprising the amino acid sequence of SEQ ID NO: 16.

A preferred antibody of the invention, the human anti-hIL-12 antibodyY61, was produced by affinity maturation of Joe 9 wild type by PCRmutagenesis of the CDR3 (as described in Example 1). Y61 had an improvedspecificity/binding affinity determined by surface plasmon resonance andby in vitro neutralization assays. The heavy and light chain CDR3s ofY61 are shown in SEQ ID NOs: 17 and 18, respectively, the heavy andlight chain CDR2s of Y61 are shown in SEQ ID NOs: 19 and 20,respectively, and the heavy and light chain CDR1s of Y61 are shown inSEQ ID NOs: 21 and 22, respectively. The VH of Y61 has the amino acidsequence of SEQ ID NO: 23 and the VL of Y61 has the amino acid sequenceof SEQ ID NO: 24 (these sequences are also shown in FIGS. 1A-1D, alignedwith Joe9).

Accordingly, in another aspect, the invention features an isolated humanantibody, or an antigen-binding portion thereof, which

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁹ M or less;

b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ IDNO: 17; and

c) has a light chain CDR3 comprising the amino acid sequence of SEQ IDNO: 18.

In a preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, has a heavy chain CDR2 comprising theamino acid sequence of SEQ ID NO: 19 and a light chain CDR2 comprisingthe amino acid sequence of SEQ ID NO: 20.

In another preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof has a heavy chain CDR1 comprising theamino acid sequence of SEQ ID NO: 21 and a light chain CDR1 comprisingthe amino acid sequence of SEQ ID NO: 22.

In yet another preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, comprising a the heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 23, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:24.

In certain embodiments, the full length antibody comprises a heavy chainconstant region, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgEconstant regions, and any allotypic variant therein as described inKabat, (Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). Preferably, the antibodyheavy chain constant region is an IgG1 heavy chain constant region.Alternatively, the antibody portion can be an Fab fragment, an F(ab′₂)fragment or a single chain Fv fragment.

Modifications of individual residues of Y61 led to the production of apanel of antibodies shown in FIGS. 2A-2H. The specificity/bindingaffinity of each antibody was determined by surface plasmon resonanceand/or by in vitro neutralization assays.

Accordingly, in another aspect, the invention features an isolated humanantibody, or an antigen-binding portion thereof, which

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁹ M or less;

b) has a heavy chain CDR3 comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NO: 404-SEQ ID NO: 469; and

c) has a light chain CDR3 comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NO: 534-SEQ ID NO: 579.

In preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, has a heavy chain CDR2 comprising theamino acid sequence selected from the group consisting of SEQ IDNO:335-SEQ ID NO: 403; and a light chain CDR2 comprising the amino acidsequence selected from the group consisting of SEQ ID NO: 506-SEQ ID NO:533.

In another preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, has a heavy chain CDR1 comprising theamino acid sequence selected from the group consisting of SEQ ID NO:288-SEQ ID NO: 334; and a light chain CDR1 comprising the amino acidsequence selected from the group consisting of SEQ ID NO: 470-SEQ ID NO:505.

In yet another preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, comprising a the heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 23, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:24.

In certain embodiments, the full length antibody comprising a heavychain constant region such as IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgEconstant regions and any allotypic variant therein as described inKabat, (Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). Preferably, the antibodyheavy chain constant region is an IgG1 heavy chain constant region.Alternatively, the antibody portion can be a Fab fragment, an F(ab′₂)fragment or a single chain Fv fragment.

A particularly preferred recombinant, neutralizing antibody of theinvention, J695, was produced by site-directed mutagenesis of contactand hypermutation amino acids residues of antibody Y61 (see Example 2and section III below). J695 differs from Y61 by a Gly to Tyrsubstitution in Y61 at position 50 of the light chain CDR2 and by a Glyto Tyr substitution at position 94 of the light chain CDR3. The heavyand light chain CDR3s of J695 are shown in SEQ ID NOs: 25 and 26,respectively, the heavy and light chain CDR2s of J695 are shown in SEQID NOs: 27 and 28, respectively, and the heavy and light chain CDR1s ofJ695 are shown in SEQ ID NOs: 29 and 30, respectively. The VH of J695has the amino acid sequence of SEQ ID NO: 31 and the VL of J695 has theamino acid sequence of SEQ ID NO: 32 (these sequences are also shown inFIGS. 1A-1D, aligned with Joe9).

Accordingly, in another aspect, the invention features an isolated humanantibody, or an antigen-binding portion thereof, which

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁹ M or less;

b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ IDNO: 25; and

c) has a light chain CDR3 comprising the amino acid sequence of SEQ IDNO: 26.

In preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, has a heavy chain CDR2 comprising theamino acid sequence of SEQ ID NO: 27, and a light chain CDR2 comprisingthe amino acid sequence of SEQ ID NO: 28.

In another preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, has a heavy chain CDR1 comprising theamino acid sequence of SEQ ID NO: 29, and a light chain CDR1 comprisingthe amino acid sequence of SEQ ID NO: 30.

In yet another preferred embodiment, the isolated human antibody, or anantigen-binding portion thereof, has a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 31, and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 32.

In certain embodiments, the full length antibody comprises a heavy chainconstant region, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgEconstant regions and any allotypic variant therein as described inKabat, (Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). Preferably, the antibodyheavy chain constant region is an IgG1 heavy chain constant region.Alternatively, the antibody portion can be an Fab fragment, an F(ab′2)fragment or a single chain Fv fragment.

Additional mutations in the preferred consensus sequences for CDR3,CDR2, and CDR1 of antibodies on the lineage from Joe 9 to J695, or fromthe lineage Y61 to J695, can be made to provide additional anti-IL-12antibodies of the invention. Such methods of modification can beperformed using standard molecular biology techniques, such as by PCRmutagenesis, targeting individual contact or hypermutation amino acidresidues in the light chain and/or heavy chain CDRs-, followed bykinetic and functional analysis of the modified antibodies as describedherein (e.g., neutralization assays described in Example 3, and byBIAcore analysis, as described in Example 5).

Accordingly, in another aspect the invention features an isolated humanantibody, or an antigen-binding portion thereof, which

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁶ M or less;

b) comprises a heavy chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 3 and a heavy chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 5, or a mutant thereof having one or more amino acidsubstitutions at a preferred selective mutagenesis position or ahypermutation position, wherein said mutant has a k_(off) rate no morethan 10-fold higher than the antibody comprising a heavy chain CDR3comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chainCDR1 comprising the amino acid sequence of SEQ ID NO: 5; and

c) comprises a light chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 2, a light chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 4, and a light chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 6, or a mutant thereof having one or more amino acidsubstitutions at a preferred selective mutagenesis position or ahypermutation position, wherein said mutant has a k_(off) rate no morethan 10-fold higher than the antibody comprising a light chain CDR3comprising the amino acid sequence of SEQ ID NO: 2, a light chain CDR2comprising the amino acid sequence of SEQ ID NO: 4, and a light chainCDR1 comprising the amino acid sequence of SEQ ID NO: 6.

In another aspect the invention features an isolated human antibody, oran antigen-binding portion thereof, which

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁹ M or less;

b) comprises a heavy chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 11 and a heavy chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 13, or a mutant thereof having one or more amino acidsubstitutions at a preferred selective mutagenesis position, contactposition or a hypermutation position, wherein said mutant has a k_(off)rate no more than 10-fold higher than the antibody comprising a heavychain CDR3 comprising the amino acid sequence of SEQ ID NO: 9, a heavychain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and aheavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13;and

c) comprises a light chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 12, and a light chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 14, or a mutant thereof having one or more amino acidsubstitutions at a preferred selective mutagenesis position, contactposition or a hypermutation position, wherein said mutant has a k_(off)rate no more than 10-fold higher than the antibody comprising a lightchain CDR3 comprising the amino acid sequence of SEQ ID NO: 10, a lightchain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and alight chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14.

An ordinarily skilled artisan will also appreciate that additionalmutations to the CDR regions of an antibody of the invention, forexample in Y61 or in J695, can be made to provide additional anti-IL-12antibodies of the invention. Such methods of modification can beperformed using standard molecular biology techniques, as describedabove. The functional and kinetic analysis of the modified antibodiescan be performed as described in Example 3 and Example 5, respectively.Modifications of individual residues of Y61 that led to theidentification of J695 are shown in FIGS. 2A-2H and are described inExample 2.

Accordingly, in another aspect the invention features an isolated humanantibody, or an antigen-binding portion thereof, which

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁹ M or less;

b) comprises a heavy chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 19 and a heavy chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 21, or a mutant thereof having one or more amino acidsubstitutions at a preferred selective mutagenesis position or ahypermutation position, wherein said mutant has a k_(off) rate no morethan 10-fold higher than the antibody comprising a heavy chain CDR3comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2comprising the amino acid sequence of SEQ ID NO: 19, and a heavy chainCDR1 comprising the amino acid sequence of SEQ ID NO: 21; and

c) comprises a light chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 18, a light chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 20, and a light chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 22, or a mutant thereof having one or more amino acidsubstitutions at a preferred selective mutagenesis position or ahypermutation position, wherein said mutant has a k_(off) rate no morethan 10-fold higher than the antibody comprising a light chain CDR3comprising the amino acid sequence of SEQ ID NO: 18, a light chain CDR2comprising the amino acid sequence of SEQ ID NO: 20, and a light chainCDR1 comprising the amino acid sequence of SEQ ID NO: 22.

In another aspect the invention features an isolated human antibody, oran antigen-binding portion thereof, which

a) inhibits phytohemagglutinin blast proliferation in an in vitro PHAassay with an IC₅₀ of 1×10⁻⁹ M or less;

b) comprises a heavy chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 27 and a heavy chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 29, or a mutant thereof having one or more amino acidsubstitutions at a preferred selective mutagenesis position or ahypermutation position, wherein said mutant has a k_(off) rate no morethan 10-fold higher than the antibody comprising a heavy chain CDR3comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2comprising the amino acid sequence of SEQ ID NO: 27, and a heavy chainCDR1 comprising the amino acid sequence of SEQ ID NO: 29; and

c) comprises a light chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 28, and a light chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 30, or a mutant thereof having one or more amino acidsubstitutions at a preferred selective mutagenesis position or ahypermutation position, wherein said mutant has a k_(off) rate no morethan 10-fold higher than the antibody comprising a light chain CDR3comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2comprising the amino acid sequence of SEQ ID NO: 28, and a light chainCDR1 comprising the amino acid sequence of SEQ ID NO: 30.

In yet another embodiment, the invention provides isolated humanantibodies, or antigen-binding portions thereof, that neutralize theactivity of human IL-12, and at least one additional primate IL-12selected from the group consisting of baboon IL-12, marmoset IL-12,chimpanzee IL-12, cynomolgus IL-12 and rhesus IL-12, but which do notneutralize the activity of the mouse IL-12.

II Selection of Recombinant Human Antibodies

Recombinant human antibodies of the invention can be isolated byscreening of a recombinant combinatorial antibody library, preferably ascFv phage display library, prepared using human VL and VH cDNAsprepared from mRNA derived from human lymphocytes. Methodologies forpreparing and screening such libraries are known in the art. In additionto commercially available kits for generating phage display libraries(e.g., the Pharmacia Recombinant Phage Antibody System, catalog no.27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no.240612), examples of methods and reagents particularly amenable for usein generating and screening antibody display libraries can be found in,for example, Kang et al. PCT Publication No. WO 92/18619; Winter et al.PCT Publication No. WO 92/20791; Breitling et al. PCT Publication No. WO93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard etal. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990)348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.

The antibody libraries used in this method are preferably scFv librariesprepared from human VL and VH cDNAs. The scFv antibody libraries arepreferably screened using recombinant human IL-12 as the antigen toselect human heavy and light chain sequences having a binding activitytoward IL-12. To select for antibodies specific for the p35 subunit ofIL-12 or the p70 heterodimer, screening assays were performed in thepresence of excess free p40 subunit. Subunit preferences can bedetermined, for example by, micro-Friguet titration, as described inExample 1.

Once initial human VL and VH segments are selected, “mix and match”experiments, in which different pairs of the selected VL and VH segmentsare screened for IL-12 binding, are performed to select preferred VL/VHpair combinations (see Example 1). Additionally, to further improve theaffinity and/or lower the off rate constant for hIL-12 binding, the VLand VH segments of the preferred VL/VH pair(s) can be randomly mutated,preferably within the CDR3 region of VH and/or VL, in a processanalogous to the in vivo somatic mutation process responsible foraffinity maturation of antibodies during a natural immune response. Thisin vitro affinity maturation can be accomplished by amplifying VH and VLregions using PCR primers complimentary to the VH CDR3 or VL CDR3,respectively, which primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode VH and VL segments into which random mutations havebeen introduced into the VH and/or VL CDR3 regions. These randomlymutated VH and VL segments can be reselected and rescreened for bindingto hIL-12 and sequences that exhibit high affinity and a low off ratefor IL-12 binding can be selected. Table 2 (see Appendix A) showsantibodies that displayed altered binding specificity/affinity producedas a result of in vitro affinity maturation.

Following selection, isolation and screening of an anti-hIL-12 antibodyof the invention from a recombinant immunoglobulin display library,nucleic acid encoding the selected antibody can be recovered from thephage particle(s) (e.g., from the phage genome) and subcloned into otherexpression vectors by standard recombinant DNA techniques. If desired,the nucleic acid can be further manipulated to create other antibodyforms of the invention (e.g., linked to nucleic acid encoding additionalimmunoglobulin domains, such as additional constant regions). To expressa recombinant human antibody isolated by screening of a combinatoriallibrary, the DNA encoding the antibody is cloned into a recombinantexpression vector and introduced into a mammalian host cells, asdescribed in further detail in Section IV below.

Methods for selecting human IL-12 binding antibodies by phage displaytechnology, and affinity maturation of selected antibodies by random orsite-directed mutagenesis of CDR regions are described in further detailin Example 1.

As described in Example 1, screening of human VL and VH cDNA librariesidentified a series of anti-IL-12 antibodies, of which the Joe 9antibody was selected for further development. A comparison of the heavychain variable region of Joe 9 with the heavy chain germline sequencesselected from the VBASE database, revealed that Joe 9 was similar to theCOS-3 germline sequence. COS-3 belongs to the V_(H)3 family of germlinesequences.

The V_(H)3 family is part of the human VH germline repertoire which isgrouped into seven families, V_(H)1-V_(H)7, based on nucleotide sequencehomology (Tomlinson et al. (1992) J. Mol. Biol., 227, 776-798 and Cooket al. (1995) Immunology Today, 16, 237-242). The V_(H)3 family containsthe highest number of members and makes the largest contribution to thegermline repertoire. For any given human V_(H)3-germline antibodysequence, the amino acid sequence identity within the entire V_(H)3family is high (See e.g., Tomlinson et al. (1992) J. Mol. Biol., 227,776-798 and Cook et al. (1995) Immunology Today, 16, 237-242). The rangeof amino acid sequence identity between any two germline VH sequences ofthe V_(H)3 family varies from 69-98 residues out of approximately 100 VHresidues, (i.e., 69-98% amino acid sequence homology between any twogermline VH sequences). For most pairs of germline sequences there is atleast 80 or more identical amino acid residues, (i.e., at least 80%amino acid sequence homology). The high degree of amino acid sequencehomology between the V_(H)3 family members results in certain amino acidresidues being present at key sites in the CDR and framework regions ofthe VH chain. These amino acid residues confer structural features uponthe CDRs.

Studies of antibody structures have shown that CDR conformations can begrouped into families of canonical CDR structures based on the key aminoacid residues that occupy certain positions in the CDR and frameworkregions. Consequently, there are similar local CDR conformations indifferent antibodies that have canonical structures with identical keyamino acid residues (Chothia et al. (1987) J. Mol. Biol., 196, 901-917and Chothia et al. (1989) Nature, 342, 877-883). Within the V_(H)3family there is a conservation of amino acid residue identity at the keysites for the CDR1 and CDR2 canonical structures (Chothia et al. (1992)J. Mol. Biol., 227, 799-817).

The COS-3 germline VH gene, is a member of the V_(H)3 family and is avariant of the 3-30 (DP-49) germline VH allele. COS-3, differs from Joe9VH amino acid sequences at only 5 positions. The high degree of aminoacid sequence homology between Joe9 VH and COS-3, and between Joe9 VHand the other V_(H)3 family members also confers a high degree of CDRstructural homology (Chothia et al. (1992) J. Mol. Biol., 227, 799-817;Chothia et al. (1987) J. Mol. Biol., 196, 901-917 and Chothia et al.(1989) Nature, 342, 877-883).

The skilled artisan will appreciate that based on the high amino acidsequence and canonical structural similarity to Joe 9, other V_(H)3family members could also be used to generate antibodies that bind tohuman IL-12. This can be performed, for example, by selecting anappropriate VL by chain-shuffling techniques (Winter et al. (1994)Annual Rev. Immunol., 12, 433-55), or by the grafting of CDRs from arodent or other human antibody including CDRs from antibodies of thisinvention onto a V_(H)3 family framework.

The human V lambda germline repertoire is grouped into 10 families basedon nucleotide sequence homology (Williams et al. (1996) J. Mol. Biol.,264, 220-232). A comparison of the light chain variable region of Joe 9with the light chain germline sequences selected from the VBASEdatabase, revealed that Joe 9 was similar to the DPL8 lambda germline.The Joe9 VL differs from DPL8 sequence at only four framework positions,and is highly homologous to the framework sequences of the other V_(λ)1family members. Based on the high amino acid sequence homology andcanonical structural similarity to Joe 9, other V_(λ)1 family membersmay also be used to generate antibodies that bind to human IL-12. Thiscan be performed, for example, by selecting an appropriate VH bychain-shuffling techniques (Winter et al. Supra, or by the grafting ofCDRs from a rodent or other human antibody including CDRs fromantibodies of this invention onto a V_(λ)1 family framework.

The methods of the invention are intended to include recombinantantibodies that bind to hIL-12, comprising a heavy chain variable regionderived from a member of the V_(H)3 family of germline sequences, and alight chain variable region derived from a member of the V_(λ)1 familyof germline sequences. Moreover, the skilled artisan will appreciatethat any member of the V_(H)3 family heavy chain sequence can becombined with any member of the V_(λ)1 family light chain sequence.

Those skilled in the art will also appreciate that DNA sequencepolymorphisms that lead to changes in the amino acid sequences of thegermline may exist within a population (e.g., the human population).Such genetic polymorphism in the germline sequences may exist amongindividuals within a population due to natural allelic variation. Suchnatural allelic variations can typically result in 1-5% variance in thenucleotide sequence of the a gene. Any and all such nucleotidevariations and resulting amino acid polymorphisms in germline sequencesthat are the result of natural allelic variation are intended to bewithin the scope of the invention.

Accordingly, in one aspect, the invention features an isolated humanantibody, or an antigen-binding portion thereof, which has the followingcharacteristics:

-   -   a) that binds to human IL-12 and dissociates from human IL-12        with a k_(off) rate constant of 0.1 s⁻¹ or less, as determined        by surface plasmon resonance, or which inhibits        phytohemagglutinin blast proliferation in an in vitro        phytohemagglutinin blast proliferation assay (PHA assay) with an        IC₅₀ of 1×10⁻⁶ M or less.    -   b) has a heavy chain variable region comprising an amino acid        sequence selected from a member of the V_(H)3 germline family,        wherein the heavy chain variable region has a mutation at a        contact or hypermutation position with an activity enhancing        amino acid residue.    -   c) has a light chain variable region comprising an amino acid        sequence selected from a member of the V_(λ)1 germline family,        wherein the light chain variable region has a mutation at a        preferred selective mutagenesis position, contact or        hypermutation position with an activity enhancing amino acid        residue.

In a preferred embodiment, the isolated human antibody, or antigenbinding has mutation in the heavy chain CDR3.

In another preferred embodiment, the isolated human antibody, or antigenbinding has mutation in the light chain CDR3.

In another preferred embodiment, the isolated human antibody, or antigenbinding has mutation in the heavy chain CDR2.

In another preferred embodiment, the isolated human antibody, or antigenbinding has mutation in the light chain CDR2.

In another preferred embodiment, the isolated human antibody, or antigenbinding has mutation in the heavy chain CDR1.

In another preferred embodiment, the isolated human antibody, or antigenbinding has mutation in the light chain CDR1.

An ordinarily skilled artisan will appreciate that based on the highamino acid sequence similarity between members of the V_(H)3 germlinefamily, or between members of the light chain V_(λ)1 germline family,that mutations to the germlines sequences can provide additionalantibodies that bind to human IL-12. Table 1 (see Appendix A) shows thegermline sequences of the V_(H)3 family members and demonstrates thesignificant sequence homology within the family members. Also shown inTable 1 are the germline sequences for V_(λ)1 family members. The heavyand light chain sequences of Joe 9 are provided as a comparison.Mutations to the germline sequences of V_(H)3 or V_(λ)1 family membersmay be made, for example, at the same amino acid positions as those madein the antibodies of the invention (e.g. mutations in Joe 9). Themodifications can be performed using standard molecular biologytechniques, such as by PCR mutagenesis, targeting individual amino acidresidues in the germline sequences, followed by kinetic and functionalanalysis of the modified antibodies as described herein (e.g.,neutralization assays described in Example 3, and by BIAcore analysis,as described in Example 5).

Accordingly, in one aspect, the invention features isolated humanantibody, or an antigen-binding portion thereof, which has the followingcharacteristics:

-   -   a) has a heavy chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:        595-667, wherein the heavy chain variable region has a mutation        at a preferred selective mutagenesis position, contact or        hypermutation position with an activity enhancing amino acid        residue.    -   b) has a light chain variable region comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:        669-675, wherein the light chain variable region has a mutation        at a preferred selective mutagenesis position, contact or        hypermutation position with an activity enhancing amino acid        residue.

An ordinarily skilled artisan will appreciate that based on the highamino acid sequence similarity between Joe 9 and COS-3 heavy chaingermline sequence, and between Joe 9 and DPL8 lambda germline sequence,that other mutations to the CDR regions of these germlines sequences canprovide additional antibodies that bind to human IL-12. Such methods ofmodification can be performed using standard molecular biologytechniques as described above.

Accordingly, in one aspect, the invention features isolated humanantibody, or an antigen-binding portion thereof, which has the followingcharacteristics:

-   -   a) that binds to human IL-12 and dissociates from human IL-12        with a k_(off) rate constant of 0.1s⁻¹ or less, as determined by        surface plasmon resonance, or which inhibits phytohemagglutinin        blast proliferation in an in vitro phytohemagglutinin blast        proliferation assay (PHA assay) with an IC₅₀ of 1×10⁻⁶ M or        less.    -   b) has a heavy chain variable region comprising the COS-3        germline amino acid sequence, wherein the heavy chain variable        region has a mutation at a preferred selective mutagenesis        position, contact or hypermutation position with an activity        enhancing amino acid residue.    -   c) has a light chain variable region comprising the DPL8        germline amino acid sequence, wherein the light chain variable        region has a mutation at a preferred selective mutagenesis        position, contact or hypermutation position with an activity        enhancing amino acid residue.

Due to certain amino acid residues occupying key sites in the CDR andframework regions in the light and heavy chain variable region,structural features are conferred at these regions. In particular, theCDR2 and CDR1 regions are subject to canonical structuralclassifications. Since there is a high degree of amino acids sequencehomology between family members, these canonical features are presentbetween family members. The skilled artisan will appreciate thatmodifications at the amino acid residues that confer these canonicalstructures would produce additional antibodies that bind to IL-12. Themodifications can be performed using standard molecular biologytechniques as described above.

Accordingly, in another aspect, the invention features an isolated humanantibody, or an antigen-binding portion thereof, which has the followingcharacteristics:

-   -   a) that binds to human IL-12 and dissociates from human IL-12        with a k_(off) rate constant of 0.1 s⁻¹ or less, as determined        by surface plasmon resonance, or which inhibits        phytohemagglutinin blast proliferation in an in vitro        phytohemagglutinin blast proliferation assay (PHA assay) with an        IC₅₀ of 1×10⁻⁶ M or less.    -   b) has a heavy chain variable region comprising an amino acid        sequence selected from a member of the V_(H)3 germline family,        wherein the heavy chain variable region comprises a CDR2 that is        structurally similar to CDR2s from other V_(H)3 germline family        members, and a CDR1 that is structurally similar to CDR1s from        other V_(H)3 germline family members, and wherein the heavy        chain variable region has a mutation at a preferred selective        mutagenesis position, contact or hypermutation position with an        activity enhancing amino acid residue;    -   c) has a light chain variable region comprising an amino acid        sequence selected from a member of the V_(λ)1 germline family,        wherein the light chain variable region comprises a CDR2 that is        structurally similar to CDR2s from other V_(λ)1 germline family        members, and a CDR1 that is structurally similar to CDR1s from        other V_(λ)1 germline family members, and wherein the light        chain variable region has a mutation at a preferred selective        mutagenesis position, contact or hypermutation position with an        activity enhancing amino acid residue.

Recombinant human antibodies of the invention have variable and constantregions which are homologous to human germline immunoglobulin sequencesselected from the VBASE database. Mutations to the recombinant humanantibodies (e.g., by random mutagenesis or PCR mutagenesis) result inamino acids that are not encoded by human germline immunoglobulinsequences. Also, libraries of recombinant antibodies which were derivedfrom human donors will contain antibody sequences that differ from theircorresponding germline sequences due to the normal process of somaticmutation that occurs during B-cell development. It should be noted thatif the “germline” sequences obtained by PCR amplification encode aminoacid differences in the framework regions from the true germlineconfiguration (i.e., differences in the amplified sequence as comparedto the true germline sequence), it may be desirable to change theseamino acid differences back to the true germline sequences (i.e.,“backmutation” of framework residues to the germline configuration).Thus, the present invention can optionally include a backmutation step.To do this, the amino acid sequences of heavy and light chain encoded bythe germline (as found as example in VBASE database) are first comparedto the mutated immunoglobulin heavy and light chain framework amino acidsequences to identify amino acid residues in the mutated immunoglobulinframework sequence that differ from the closest germline sequences.Then, the appropriate nucleotides of the mutated immunoglobulin sequenceare mutated back to correspond to the germline sequence, using thegenetic code to determine which nucleotide changes should be made.Mutagenesis of the mutated immunoglobulin framework sequence is carriedout by standard methods, such as PCR-mediated mutagenesis (in which themutated nucleotides are incorporated into the PCR primers such that thePCR product contains the mutations) or site-directed mutagenesis. Therole of each amino acid identified as candidate for backmutation shouldbe investigated for a direct or indirect role in antigen binding and anyamino acid found after mutation to affect any desirable characteristicof the human antibody should not be included in the final humanantibody; as an example, activity enhancing amino acids identified bythe selective mutagenesis approach will not be subject to backmutation.Assays to determine the characteristics of the antibody resulting frommutagenesis can include ELISA, competitive ELISA, in vitro and in vivoneutralization assays and/or (see e.g. Example 3) immunohistochemistrywith tissue sections from various sources (including human, primateand/or other species).

To minimize the number of amino acids subject to backmutation thoseamino acid positions found to be different from the closest germlinesequence but identical to the corresponding amino acid in a secondgermline sequence can remain, provided that the second germline sequenceis identical and colinear to the sequence of the human antibody of theinvention for at least 10, preferably 12 amino acids, on both sides ofthe amino acid in question. This would assure that any peptide epitopepresented to the immune system by professional antigen presenting cellsin a subject treated with the human antibody of the invention would notbe foreign but identical to a self-antigen, i.e. the immunoglobulinencoded by that second germline sequence. Backmutation may occur at anystage of antibody optimization; preferably, backmutation occurs directlybefore or after the selective mutagenesis approach. More preferably,backmutation occurs directly before the selective mutagenesis approach.

III. Modifications to Preferred Selective Mutagenesis Positions, Contactand/or Hypermutation Positions

Typically, selection of antibodies with improved affinities can becarried out using phage display methods, as described in section IIabove. This can be accomplished by randomly mutating combinations of CDRresidues and generating large libraries containing antibodies ofdifferent sequences. However, for these selection methods to work, theantibody-antigen reaction must tend to equilibrium to allow, over time,preferential binding of higher affinity antibodies to the antigen.Selection conditions that would allow equilibrium to be establishedcould not be determined (presumably due to additional non-specificinteractions between the antigen and phage particle) when phage displaymethods were used to improve the affinity of selected anti-IL-12antibodies, upon attaining a certain level of affinity achieved (i.e.,that of antibody Y61). Accordingly, antibodies with even higheraffinities could not be selected by phage display methods. Thus, for atleast certain antibodies or antigens, phage display methods are limitingin their ability to select antibodies with a highly improved bindingspecificity/affinity. Accordingly, a method termed Selective MutagenesisApproach which does not require phage display affinity maturation ofantibodies, was established to overcome this limitation and is providedby the invention. Although this Selective Mutagenesis Approach wasdeveloped to overcome limitations using the phage display system, itshould be noted that this method can also be used with the phage displaysystem. Moreover, the selective mutagenesis approach can be used toimprove the activity of any antibody.

To improve the activity (e.g., affinity or neutralizing activity) of anantibody, ideally one would like to mutate every CDR position in boththe heavy and light chains to every other possible amino acid residue.However, since there are, on average, 70 CDR positions within anantibody, such an approach would be very time consuming and laborintensive. Accordingly, the method of the invention allows one toimprove the activity of the antibody by mutating only certain selectedresidues within the heavy and/or light chain CDRs. Furthermore, themethod of the invention allows improvement in activity of the antibodywithout affecting other desirable properties of the antibody.

Determining which amino acid residues of an antibody variable region arein contact with an antigen cannot be accurately predicted based onprimary sequence or their positions within the variable region.Nevertheless, alignments of sequences from antibodies with differentspecificities conducted by Kabat et al. have identified the CDRs aslocal regions within the variable regions which differ significantlyamong antibodies (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-393,Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242). Structural studies have shown that theantigen binding surface is formed by amino acid residues present in theCDRs. Other amino acid residues outside the CDR are also known to playstructural roles or be directly involved in antigen binding. Therefore,for each antigen-antibody pair, amino acid residues within and outsideof the CDRs may be important.

The sequence alignment studies by Tomlison et al identified a number ofpositions in the heavy and light chain CDR1 and CDR2, and in a portionof the kappa chain CDR3 which are frequent sites of somatic mutation.(Tomlison et al (1996) J. Mol. Biol. 256: 813-817). In particular,positions H31, H31B, H33, H33B, H52B, H56, H58, L30, L31, L31A, L50,L53, L91, L92, L93 and L94 were identified as frequent sites for somaticmutation. However, this analysis excludes the important heavy chain CDR3regions, and sections of the light chain CDR3 which are known to lie inthe center of an antibody binding site, and potentially provideimportant interactions with an antigen. Furthermore, Tomlison et al.propose that somatic diversity alone does not necessarily predict a roleof a specific amino acid in antigen binding, and suggest conserved aminoacid residues that contact the antigen, and diverse amino acid residueswhich do not contact the antigen. This conclusion is further supportedby mutational studies on the role of somatic mutations to antibodyaffinity (Sharon, (1990), PNAS, 87:4814-7). Nineteen somatic mutationsin a high-affinity anti-p-azophenylarsonate (Ars) antibody weresimultaneously replaced with their corresponding germline residues,generating a germline version of the anti-Ars antibody which had atwo-hundred fold loss in activity. The full affinity of the anti-Arsantibody could be recovered by restoring only three of the nineteensomatic mutations, demonstrating that many somatic mutations may bepermitted that do not contribute to antigen binding activity.

The result can be explained in part by the nature of antibody diversityitself. Immature B-cells may produce initially low affinity antibodiesthat recognize a number of self or non-self antigens. Moreover,antibodies may undergo in the course of affinity maturation sequencevariations that may cause self-reactivity. Hypermutation of such lowaffinity antibodies may serve to abolish self-reactivity (“negativeselection”) and increase affinity for the foreign antigen. Therefore,the analysis of primary and structural data of a large number ofantibodies does not provide a method of predicting either (1) the roleof somatic hyper-mutation sites in the affinity maturation processversus the process of decreasing affinity towards unwanted antigens, or(2) how a given amino acid contributes to the properties of a specificantigen-antibody pair.

Other attempts to address the role of specific amino acid residues inantigen recognition were made by analyzing a number of crystalstructures of antigen-antibody complexes (MacCallum et al. (1996) J.Mol. Biol. 262: 732-745). The potential role of positions located withinand outside the CDRs was indicated. Positions in CDRs involved inantigen binding in more than 10 of 26 analyzed structures included H31,H33, H50, H52, H53, H54, H56, H58, H95, H96, H97, H98 and H100 in theheavy chain and L30A, L32, L91, L92, L93, L94, L96 in the light chain.However, the authors noted that prediction of antigen contacts usingthese and other structural data may over and under predict contactpositions, leading to the speculation that a different strategy may haveto be applied to different antigens.

Pini et al. describe randomizing multiple residues in antibody CDRsequences in a large phage display library to rapidly increase antibodyaffinity (Pini et al. (1998) J. Biol Chem. 273: 21769-21776). However,the high affinity antibodies discussed by Pini et al. had mutations in atotal of eight positions, and a reductionary analysis of which changesare absolutely required to improve affinity of the antibody becomesimpractical because of the large number of possible combinations to betested for the smallest number of amino acids required.

Furthermore, randomizing multiple residues may not necessarily preserveother desired properties of the antibody. Desirable properties orcharacteristics of an antibody are art-recognized and include forexample, preservation of non-cross reactivity, e.g., with other proteinsor human tissues and preservation of antibody sequences that are closeto human germline immunoglobulin sequences improvement of neutralizationpotency. Other desirable properties or characteristics include abilityto preserve species cross reactivity, ability to preserve epitopespecificity and ability to preserve high expression levels of protein inmammalian cells. The desirable properties or characteristics can beobserved or measured using art-recognized techniques including but notlimited to ELISA, competitive ELISA, in vitro and in vivo neutralizationassays (see e.g. Example 3), immunohistochemistry with tissue sectionsfrom different sources including human, primate or other sources as theneed may be, and studies to expression in mammalian cells usingtransient expression or stable expression.

In addition, the method of Pini et al may introduce more changes thanthe minimal number actually required to improve affinity and may lead tothe antibodies triggering anti-human-antibody (HAMA) formation in humansubjects. Further, as discussed elsewhere, the phage display asdemonstrated here, or other related method including ribosome displaymay not work appropriately upon reaching certain affinities betweenantibody and antigen and the conditions required to reach equilibriummay not be established in a reasonable time frame because of additionalinteractions including interactions with other phage or ribosomecomponents and the antigen.

The ordinarily skilled artisan may glean interesting scientificinformation on the origin of antibody diversity from the teachings ofthe references discussed above. The present invention, however, providesa method for increasing antibody affinity of a specific antigen-antibodypair while preserving other relevant features or desirablecharacteristics of the antibody. This is especially important whenconsidering the desirability of imparting a multitude of differentcharacteristics on a specific antibody including antigen binding.

If the starting antibody has desirable properties or characteristicswhich need to be retained, a selective mutagenesis approach can be thebest strategy for preserving these desirable properties while improvingthe activity of the antibody. For example, in the mutagenesis of Y61,the aim was to increase affinity for hIL-12, and to improve theneutralization potency of the antibody while preserving desiredproperties. Desired properties of Y61 included (1) preservation ofnon-cross reactivity with other proteins or human tissues, (2)preservation of fine epitope specificity, i.e. recognizing a p40 epitopepreferably in the context of the p70 (p40/p35) heterodimer, therebypreventing binding interference from free soluble p40; and (3)generation of an antibody with heavy and light chain amino acidsequences that were as close as possible to their respective germlineimmunoglobulin sequences.

In one embodiment, the method of the invention provides a selectivemutagenesis approach as a strategy for preserving the desirableproperties or characteristics of the antibody while improving theaffinity and/or neutralization potency. The term “selective mutagenesisapproach” is as defined above and includes a method of individuallymutating selected amino acid residues. The amino acid residues to bemutated may first be selected from preferred selective mutagenesispositions, then from contact positions, and then from hypermutationpositions. The individual selected position can be mutated to at leasttwo other amino acid residue and the effect of the mutation both on thedesired properties of the antibody, and improvement in antibody activityis determined.

The Selective Mutagenesis approach comprises the steps of:

selecting candidate positions in the order 1) preferred selectivemutagenesis positions; 2) contact positions; 3) hypermutation positionsand ranking the positions based on the location of the position withinthe heavy and light chain variable regions of an antibody (CDR3preferred over CDR2 preferred over CDR1);

individually mutating candidate preferred selective mutagenesispositions, hypermutation and/or contact positions in the order ofranking, to all possible other amino acid residues and analyzing theeffect of the individual mutations on the activity of the antibody inorder to determine activity enhancing amino acid residues;

if necessary, making stepwise combinations of the individual activityenhancing amino acid residues and analyzing the effect of the variouscombinations on the activity of the antibodies; selecting mutantantibodies with activity enhancing amino acid residues and ranking themutant antibodies based on the location and identity of the amino acidsubstitutions with regard to their immunogenic potential. Highestranking is given to mutant antibodies that comprise an amino acidsequence which nearly identical to a variable region sequence that isdescribed in a germline database, or has an amino acid sequence that iscomparable to other human antibodies. Lower ranking is given to mutantantibodies containing an amino acid substitution that is rarelyencountered in either germline sequences or the sequences of other humanantibodies. The lowest ranking is given to mutant antibodies with anamino acid substitution that has not been encountered in a germlinesequence or the sequence of another human antibody. As set forth above,mutant antibodies comprising at least one activity enhancing amino acidresidue located in CDR3 is preferred over CDR2 which is preferred overCDR1. The CDRs of the heavy chain variable regions are preferred overthose of the light chain variable region.

The mutant antibodies can also be studied for improvement in activity,e.g. when compared to their corresponding parental antibody. Theimprovement in activity of the mutant antibody can be determined forexample, by neutralization assays, or binding specificity/affinity bysurface plasmon resonance analysis (see Example 3). Preferably, theimprovement in activity can be at least 2-20 fold higher than theparental antibody. The improvement in activity can be at least “x₁” to“x₂” fold higher than the parental antibody wherein “x₁” and “x₂” areintegers between and including 2 to 20, including ranges within thestate range, e.g. 2-15, e.g. 5-10.

The mutant antibodies with the activity enhancing amino acid residuealso can be studied to determine whether at least one other desirableproperty has been retained after mutation. For example, with anti-hIL-12antibodies testing for, (1) preservation of non-cross reactivity withother proteins or human tissues, (2) preservation of epitoperecognition, i.e. recognizing a p40 epitope preferably in the context ofthe p70 (p40/p35) heterodimer, thereby preventing binding interferencefrom free soluble p40; and (3) generation of antibodies with heavy andlight chain amino acid sequences that were as close as possible to theirrespective germline immunoglobulin sequences, and determining whichwould be least likely to elicit a human immune response based on thenumber of differences from the germline sequence. The same observationscan be made on an antibody having more than one activity enhancing aminoacid residues, e.g. at least two or at least three activity enhancingamino acid residues, to determine whether retention of the desirableproperty or characteristic has occurred.

An example of the use of a “selective mutagenesis approach”, in themutagenesis of Y61 is described below. The individual mutations H31S→E,L50→Y, or L94G→Y each improved neutralization activity of the antibody.However, when combination clones were tested, the activity of thecombined clone H31S→E+L50→Y+L94G→Y was no better than L50→Y+L94G→Y(J695). Therefore, changing the germline amino acid residue Ser to Gluat position 31 of CDR1 was unnecessary for the improved activity of J695over Y61. The selective mutagenesis approach therefore, identified theminimal number of changes that contributed to the final activity,thereby reducing the immunogenic potential of the final antibody andpreserving other desired properties of the antibody.

Isolated DNA encoding the VH and VL produced by the selected mutagenesisapproach can be converted into full length antibody chain genes, to Fabfragment genes as to a scFV gene, as described in section IV. Forexpression of VH and VL regions produced by the selected mutagenesisapproach, expression vectors encoding the heavy and light chain can betransfected into variety host cells as described in detail in sectionIV. Preferred host cells include either prokaryotic host cells, forexample, E. coli, or eukaryotic host cells, for example, yeast cells,e.g., S. cerevisae. Most preferred eukaryotic host cells are mammalianhost cells, described in detail in section IV.

The selective mutagenesis approach provides a method of producingantibodies with improved activities without prior affinity maturation ofthe antibody by other means. The selective mutagenesis approach providesa method of producing antibodies with improved affinities which havebeen subject to back mutations. The selective mutagenesis approach alsoprovides a method of improving the activity of affinity maturedantibodies.

The skilled artisan will recognize that the selective mutagenesisapproach can be used in standard antibody manipulation techniques knownin the art. Examples include, but are not limited to, CDR graftedantibodies, chimeric antibodies, scFV fragments, Fab fragments of a fulllength antibodies and human antibodies from other sources, e.g.,transgenic mice.

Rapid large scale mutational analysis of antibodies include in vitrotranscription and translation using ribosome display technology (seee.g., Hanes et al., (1997) Proc. Natl. Acad. Sci. 94: 4937-4942; DallAcqua et al., (1998) Curr. Opin. Struc. Biol. 8: 443-450; He et al.,(1997) Nucleic Acid Res. 25: 5132-5134), and U.S. Pat. Nos. 5,643,768and 5,658,754 issued to Kawasaki. The selective mutagenesis approachalso provides a method of producing antibodies with improved activitiesthat can be selected using ribosomal display techniques.

In the methods of the invention, antibodies or antigen binding portionsthereof are further modified by altering individual positions in theCDRs of the HCVR and/or LCVR. Although these modifications can be madein phage-displayed antibodies, the method is advantageous in that it canbe performed with antibodies that are expressed in other types of hostsystems, such as bacterial, yeast or mammalian cell expression systems.The individual positions within the CDRs selected for modification arebased on the positions being a contact and/or hypermutation position.

Preferred contact positions and hypermutation positions as definedherein are shown in Table 3 (see Appendix A) and their modification inaccordance with the method of the invention is described in detail inExample 2. Preferred contact positions are selected from the groupconsisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54,H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53,L55, L91, L92, L93, L94 and L96. Preferred hypermutation positions areselected from the group consisting of H30, H31, H31B, H32, H52, H56,H58, L30, L31, L32, L53 and L93. More preferred amino acid residues(referred to as “preferred selective mutagenesis positions”) are bothcontact and hypermutation positions and are selected from the groupconsisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32,L50, L91, L92, L93, L94. Particularly preferred contact positions areselected from the group consisting of L50 and L94.

Preferred activity enhancing amino acid residues replace amino acidresidues located at positions selected from the group consisting of H30,H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96,H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,L94, and L96. More preferred activity enhancing amino acid residuesreplace amino acid residues located at positions H30, H31, H31B, H32,H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94.Particularly, preferred activity enhancing amino acid residues replaceamino acid residues located at positions selected from the groupconsisting of L50 and L94.

In general, the method of the invention involves selecting a particularpreferred selective mutagenesis position, contact and/or hypermutationposition within a CDR of the heavy or light chain of a parent antibodyof interest, or antigen binding portion thereof, randomly mutagenizingthat individual position (e.g., by genetic means using a mutagenicoligonucleotide to generate a “mini-library” of modified antibodies), ormutating a position to specific desired amino acids, to identifyactivity enhancing amino acid residues expressing, and purifying themodified antibodies (e.g., in a non-phage display host system),measuring the activity of the modified antibodies for antigen (e.g., bymeasuring k_(off) rates by BIAcore analysis), repeating these steps forother CDR positions, as necessary, and combining individual mutationsshown to have improved activity and testing whether the combination(s)generate an antibody with even greater activity (e.g., affinity orneutralizing potency) than the parent antibody, or antigen-bindingportion thereof.

Accordingly, in one embodiment, the invention provides a method forimproving the activity of an antibody, or antigen-binding portionthereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof;

b) selecting in order a 1) preferred selective mutagenesis position, 2)contact position, or 3) hypermutation position within a complementaritydetermining region (CDR) for mutation, thereby identifying a selectedpreferred selective mutagenesis position, contact or hypermutationposition;

c) individually mutating said selected preferred selective mutagenesisposition, contact or hypermutation position to at least two other aminoacid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof;

e) optionally, repeating steps a) through d) for at least one otherpreferred selective mutagenesis position, contact or hypermutationposition;

f) combining, in the parent antibody, or antigen-binding portionthereof, individual mutations shown to have improved activity, to formcombination antibodies, or antigen-binding portions thereof; and

g) evaluating the activity of the combination antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof;

until an antibody, or antigen-binding portion thereof, with an improvedactivity, relative to the parent antibody, or antigen-binding portionthereof, is obtained. Preferably, the selected antibody or antibodieshave an improved activity without loss or with retention of at least onedesirable characteristic or property of the parental antibody asdescribed above. The desirable characteristic or property can bemeasured or observed by the ordinarily skilled artisan usingart-recognized techniques.

Preferred contact positions are selected from the group consisting ofH30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95,H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92,L93, L94 and L96. Preferred hypermutation positions are selected fromthe group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31,L32, L53 and L93. More preferred selective mutagenesis positions areselected from the group consisting of H30, H31, H31B, H32, H33, H52,H56, H58, L30, L31, L32, L50, L91, L92, L93 and L94. Particularlypreferred contact positions are selected from the group consisting ofL50 and L94.

In another embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof;

b) selecting a preferred selective mutagenesis position, contact orhypermutation position within a complementarity determining region (CDR)for mutation;

c) individually mutating said selected preferred selective mutagenesisposition, contact or hypermutation position to at least two other aminoacid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof, thereby identifying an activityenhancing amino acid residue;

e) optionally, repeating steps a) through d) for at least one otherpreferred selective mutagenesis position, contact or hypermutationposition;

f) combining, in the parent antibody, or antigen-binding portionthereof, two individual activity enhancing amino acid residues shown tohave improved activity, to form combination antibodies, orantigen-binding portions thereof; and

g) evaluating the activity of the combination antibodies, orantigen-binding portions thereof with two activity enhancing amino acidresidues, relative to the parent antibody or antigen-binding portionthereof;

until an antibody, or antigen-binding portion thereof, with an improvedactivity, relative to the parent antibody, or antigen-binding portionthereof, is obtained.

Preferred contact positions are selected from the group consisting ofH30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95,H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92,L93, L94 and L96. Preferred hypermutation positions are selected fromthe group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31,L32, L53 and L93. More preferred selective mutagenesis positions areselected from the group consisting of H30, H31, H31B, H32, H33, H52,H56, H58, L30, L31, L32, L50, L91, L92, L93 and L94. Particularlypreferred contact positions are selected from the group consisting ofL50 and L94.

In another embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof;

b) selecting a preferred selective mutagenesis position, contact orhypermutation position within a complementarity determining region (CDR)for mutation;

c) individually mutating said selected preferred selective mutagenesisposition, contact or hypermutation position to at least two other aminoacid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof, thereby identifying an activityenhancing amino acid residue;

e) optionally, repeating steps a) through d) for at least one otherpreferred selective mutagenesis position, contact or hypermutationposition;

f) combining, in the parent antibody, or antigen-binding portionthereof, three individual activity enhancing amino acid residues shownto have improved activity, to form combination antibodies, orantigen-binding portions thereof; and

g) evaluating the activity of the combination antibodies, orantigen-binding portions thereof with two activity enhancing amino acidresidues, relative to the parent antibody or antigen-binding portionthereof;

until an antibody, or antigen-binding portion thereof, with an improvedactivity, relative to the parent antibody, or antigen-binding portionthereof, is obtained.

Preferably, the activity enhancing amino acid residue replaces aminoacid residues located at positions selected from the group consisting ofH30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95,H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92,L93, L94 and L96.

Following mutagenesis of individual selected positions, mutated clonescan be sequenced to identify which amino acid residues have beenintroduced into the selected position in each clone. A small number ofclones (e.g., about 24) can be selected for sequencing, whichstatistically should yield 10-15 unique antibodies, whereas largernumbers of clones (e.g., greater than 60) can be sequenced to ensurethat antibodies with every possible substitution at the selectedposition are identified.

In one embodiment, contact and/or hypermutation positions within theCDR3 regions of the heavy and/or light chains are first selected formutagenesis. However, for antibodies that have already been affinitymatured in vitro by random mutagenesis of the CDR3 regions via phagedisplay selection, it may be preferably to first select contact and/orhypermutation positions within CDR1 or CDR2 of the heavy and/or lightchain.

In a more preferred embodiment, preferred selective mutagenesispositions within the CDR3 regions of the heavy and/or light chains arefirst selected for mutagenesis. However, for antibodies that havealready been affinity matured in vitro by random mutagenesis of the CDR3regions via phage display selection, it may be preferably to firstselect preferred selective mutagenesis positions within CDR1 or CDR2 ofthe heavy and/or light chain.

In another preferred embodiment, the optimization of a selected antibodyby the selective mutagenesis approach is done sequentially as follows:preferred selective mutagenesis positions selected from the groupconsisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32,L50, L91, L92, L93, L94 are mutated first to at least 2 other aminoacids each (preferably 5-14 other amino acids) and the resultingantibodies are characterized for increased affinity, neutralizationpotency (and possibly also for at least one other retainedcharacteristic or property discussed elsewhere). If a mutation of asingle preferred selective mutagenesis position does not increase theaffinity or neutralization potency at all or sufficiently and if eventhe combination of multiple activity enhancing amino acids replacingamino acids in preferred selective mutagenesis positions does not resultin an combination antibody which meets the target activity (includingaffinity and/or neutralization potency), additional amino acid residueswill be selected for selective mutagenesis from the group consisting ofH35, H50, H53, H54, H95, H96, H97, H98, L30A and L96 are mutated to atleast 2 other amino acids each (preferably 5-14 other amino acids) andthe resulting antibodies are characterized for increased affinity,neutralization potency (and possibly also for at least one otherretained characteristic or property discussed elsewhere).

If a mutation of a single amino acid residue selected from the groupconsisting of H35, H50, H53, H54, H95, H96, H97, H98, L30A and L96 doesnot increase the activity (including affinity and/or neutralizationpotency) at all or not sufficiently and if even the combination ofmultiple activity enhancing amino acids replacing amino acids in thosepositions does not result in an combination antibody which meets thetargeted activity (including affinity and/or target neutralizationpotency), additional amino acid residues will be selected for selectivemutagenesis from the group consisting of H33B, H52B, L31A and aremutated to at least 2 other amino acids each (preferably 5-14 otheramino acids) and the resulting antibodies are characterized forincreased affinity, neutralization potency (and possibly also for atleast one other retained characteristic or property discussedelsewhere).

It should be understood that the sequential selective mutagenesisapproach may end at any of the steps outline above as soon as anantibody with the desired activity (including affinity andneutralization potency) has been identified. If mutagenesis of thepreselected positions has identified activity enhancing amino acidsresidues but the combination antibody still do not meet the targets setfor activity (including affinity and neutralization potency) and/or ifthe identified activity enhancing amino acids also affect other desiredcharacteristics and are therefore not acceptable, the remaining CDRresidues may be subjected to mutagenesis (see section IV).

The method of the invention can be used to improve activity of anantibody, or antigen binding portion thereof, to reach a predeterminedtarget activity (e.g. a predetermined affinity and/or neutralizationpotency, and/or a desired property or characteristic).

Accordingly, the invention provides a method of improving the activityof an antibody, or antigen-binding portion thereof, to attain apredetermined target activity, comprising:

a) providing a parent antibody a antigen-binding portion thereof;

b) selecting a preferred selective mutagenesis position selected fromgroup consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,L32, L50, L91, L92, L93, L94.

c) individually mutating the selected preferred selective mutagenesisposition to at least two other amino acid residues to hereby create afirst panel of mutated antibodies, or antigen binding portions thereof;

d) evaluating the activity of the first panel of mutated antibodies, orantigen binding portions thereof to determined if mutation of a singleselective mutagenesis position produces an antibody or antigen bindingportion thereof with the predetermined target activity or a partialtarget activity;

e) combining in a stepwise fashion, in the parent antibody, or antigenbinding portion thereof, individual mutations shown to have an improvedactivity, to form combination antibodies, or antigen binding portionsthereof.

f) evaluating the activity of the combination antibodies, or antigenbinding portions thereof to determined if the combination antibodies, orantigen binding portions thereof have the predetermined target activityor a partial target activity.

g) if steps d) or f) do not result in an antibody or antigen bindingportion thereof having the predetermined target activity, or result anantibody with only a partial activity, additional amino acid residuesselected from the group consisting of H35, H50, H53, H54, H95, H96, H97,H98, L30A and L96 are mutated to at least two other amino acid residuesto thereby create a second panel of mutated antibodies orantigen-binding portions thereof;

h) evaluating the activity of the second panel of mutated antibodies orantigen binding portions thereof, to determined if mutation of a singleamino acid residue selected from the group consisting of H35, H50, H53,H54, H95, H96, H97, H98, L30A and L96 results an antibody or antigenbinding portion thereof, having the predetermined target activity or apartial activity;

i) combining in stepwise fashion in the parent antibody, orantigen-binding portion thereof, individual mutations of step g) shownto have an improved activity, to form combination antibodies, or antigenbinding portions thereof;

j) evaluating the activity of the combination antibodies or antigenbinding portions thereof, to determined if the combination antibodies,or antigen binding portions thereof have the predetermined targetactivity or a partial target activity;

k) if steps h) or j) do not result in an antibody or antigen bindingportion thereof having the predetermined target activity, or result inan antibody with only a partial activity, additional amino acid residuesselected from the group consisting of H33B, H52B and L31A are mutated toat least two other amino acid residues to thereby create a third panelof mutated antibodies or antigen binding portions thereof;

l) evaluating the activity of the third panel of mutated antibodies orantigen binding portions thereof, to determine if a mutation of a singleamino acid residue selected from the group consisting of H33B, H52B andL31A resulted in an antibody or antigen binding portion thereof, havingthe predetermined target activity or a partial activity;

m) combining in a stepwise fashion in the parent antibody, or antigenbinding portion thereof, individual mutation of step k) shown to have animproved activity, to form combination antibodies, or antigen bindingportions, thereof;

n) evaluating the activity of the combination antibodies orantigen-binding portions thereof, to determine if the combinationantibodies, or antigen binding portions thereof have the predeterminedtarget activity to thereby produce an antibody or antigen bindingportion thereof with a predetermined target activity.

A number of mutagenesis methods can be used, including PCR assembly,Kunkel (dut-ung-) and thiophosphate (Amersham Sculptor kit)oligonucleotide-directed mutagenesis.

A wide variety of host expression systems can be used to express themutated antibodies, including bacterial, yeast, baculoviral andmammalian expression systems (as well as phage display expressionsystems). An example of a suitable bacterial expression vector ispUC119(Sfi). Other antibody expression systems are known in the artand/or are described below in section IV.

The modified antibodies, or antigen binding portions thereof, producedby the method of the invention can be identified without the reliance onphage display methods for selection. Accordingly, the method of theinvention is particularly advantageous for improving the activity of arecombinant parent antibody or antigen-binding portion thereof, that wasobtained by selection in a phage-display system but whose activitycannot be further improved by mutagenesis in the phage-display system.

Accordingly, in another embodiment, the invention provides a method forimproving the affinity of an antibody, or antigen-binding portionthereof, comprising:

a) providing a recombinant parent antibody or antigen-binding portionthereof; that was obtained by selection in a phage-display system butwhose activity cannot be further improved by mutagenesis in saidphage-display system;

b) selecting a preferred selective mutagenesis position, contact orhypermutation position within a complementarity determining region (CDR)for mutation, thereby identifying a selected contact or hypermutationposition;

c) individually mutating said selected preferred selective mutagenesisposition, contact or hypermutation position to at least two other aminoacid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof, and expressing said panel in anon-phage display system;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof;

e) optionally repeating steps b) through d) for at least one otherpreferred selective mutagenesis position, contact or hypermutationposition;

f) combining, in the parent antibody, or antigen-binding portionthereof, individual mutations shown to have improved activity, to formcombination antibodies, or antigen-binding portions thereof; and

g) evaluating the activity of the combination antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof; until an antibody, or antigen-bindingportion thereof, with an improved activity, relative to the parentantibody, or antigen-binding portion thereof, is obtained.

Preferred contact positions are selected from the group consisting ofH30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95,H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92,L93, L94 and L96. Preferred hypermutation positions are selected fromthe group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31,L32, L53 and L93. More preferred selective mutagenesis positions areselected from the group consisting of H30, H31, H31B, H32, H33, H52,H56, H58, L30, L31, L32, L50, L91, L92, L93 and L94. Particularlypreferred contact positions are selected from the group consisting ofL50 and L94.

With available methods it is not possible or it is extremely laboriousto derive an antibody with increased binding affinity and neutralizationpotency while retaining other properties or characteristics of theantibodies as discussed above. The method of this invention, however,can readily identify such antibodies. The antibodies subjected to themethod of this invention can come from any source.

Therefore, in another embodiment, the invention provides a method forimproving the activity of an antibody, or antigen-binding portionthereof, comprising:

a) providing a recombinant parent antibody or antigen-binding portionthereof;

b) selecting a preferred selective mutagenesis position, contact orhypermutation position within a complementarity determining region (CDR)for mutation, thereby identifying a selected preferred selectivemutagenesis position, contact or hypermutation position;

c) individually mutating said selected preferred selective mutagenesisposition, contact or hypermutation position to at least two other aminoacid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof and expressing said panel in anappropriate expression system;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof, thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof for at least one other property or characteristics,wherein the property or characteristic is one that needs to be retainedin the antibody;

until an antibody, or antigen-binding portion thereof, with an improvedactivity and at least one retained property or characteristic, relativeto the parent antibody, or antigen-binding portion thereof, is obtained.

In a preferred embodiment, the contact positions are selected from thegroup consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52,L53, L55, L91, L92, L93, L94 and L96 and the other characteristic isselected from 1) preservation of non-crossreactivity with other proteinsor human tissues, 2) preservation of epitope recognition, i.e.recognizing p40 epitope preferably in the context of the p70 p40/p35heterodimer preventing binding interference from free, soluble p40and/or 3) to produce an antibody with a close to germline immunoglobulinsequence.

In another preferred embodiment, the hypermutation positions areselected from the group consisting of H30, H31, H31B, H32, H52, H56,H58, L30, L31, L32, L53 and L93 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence.

In a more preferred embodiment the residues for selective mutagenesisare selected from the preferred selective mutagenesis positions from thegroup consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,L32, L50, L91, L92, L93, L94 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence.

In a more preferred embodiment, the contact positions are selected fromthe group consisting of L50 and L94 and the other characteristic isselected from 1) preservation of non-crossreactivity with other proteinsor human tissues, 2) preservation of epitope recognition, i.e.recognizing p40 epitope preferably in the context of the p70 p40/p35heterodimer preventing binding interference from free, soluble p40and/or 3) to produce an antibody with a close to germline immunoglobulinsequence.

If therefore, the affinity of an antibody for a specific antigen shouldbe improved, but where the phage display (or related system includingribosome display) method is no longer applicable, and other desirableproperties or characteristics should be retained, the method of theinvention can be used. Accordingly, in another embodiment, the inventionprovides a method for improving the activity of an antibody, orantigen-binding portion thereof, comprising:

a) providing a recombinant parent antibody or antigen-binding portionthereof; that was obtained by selection in a phage-display system butwhose activity cannot be further improved by mutagenesis in saidphage-display system;

b) selecting a preferred selective mutagenesis position, contact orhypermutation position within a complementarity determining region (CDR)for mutation, thereby identifying a selected preferred selectivemutagenesis position, contact or hypermutation position;

c) individually mutating said selected preferred selective mutagenesisposition, contact or hypermutation position to at least two other aminoacid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof, and expressing said panel in anon-phage display system;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof for at least one other property or characteristic,wherein the property or characteristic is one that needs to be retained,until an antibody, or antigen-binding portion thereof, with an improvedactivity and at least one retained property or characteristic, relativeto the parent antibody, or antigen-binding portion thereof, is obtained.

f) optionally, repeating steps a) through e) for at least one otherpreferred selective mutagenesis position, contact or hypermutationposition;

g) combining, in the parent antibody, or antigen-binding portionthereof, at least two individual activity enhancing amino acid residuesshown to have improved activity and at least one retained property orcharacteristic, to form combination antibodies, or antigen-bindingportions thereof; and

h) evaluating the activity of the combination antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof; until an antibody, or antigen-bindingportion thereof, with an improved activity and at least one retainedother property or characteristic, relative to the parent antibody, orantigen-binding portion thereof, is obtained.

In a preferred embodiment, the contact positions are selected from thegroup consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52,L53, L55, L91, L92, L93, L94 and L96 and the other characteristic isselected from 1) preservation of non-crossreactivity with other proteinsor human tissues, 2) preservation of epitope recognition, i.e.recognizing p40 epitope preferably in the context of the p70 p40/p35heterodimer preventing binding interference from free, soluble p40and/or 3) to produce an antibody with a close to germline immunoglobulinsequence.

In another preferred embodiment, the hypermutation positions areselected from the group consisting of H30, H31, H31B, H32, H52, H56,H58, L30, L31, L32, L53 and L93 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence.

In a more preferred embodiment the residues for selective mutagenesisare selected from the preferred selective mutagenesis positions from thegroup consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,L32, L50, L91, L92, L93, L94 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence.

In a more preferred embodiment, the contact positions are selected fromthe group consisting of L50 and L94 and the other characteristic isselected from 1) preservation of non-crossreactivity with other proteinsor human tissues, 2) preservation of epitope recognition, i.e.recognizing p40 epitope preferably in the context of the p70 p40/p35heterodimer preventing binding interference from free, soluble p40and/or 3) to produce an antibody with a close to germline immunoglobulinsequence.

In another embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a recombinant parent antibody or antigen-binding portionthereof; that was obtained by selection in a phage-display system butwhose activity cannot be further improved by mutagenesis in saidphage-display system;

b) selecting a preferred selective mutagenesis position, contact orhypermutation position within a complementarity determining region (CDR)for mutation, thereby identifying a selected contact or hypermutationposition;

c) individually mutating said selected preferred selective mutagenesisposition, contact or hypermutation position to at least two other aminoacid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof, and expressing said panel in anon-phage display system;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof for at least one other property or characteristic,wherein the property or characteristic is one that needs to be retained,until an antibody, or antigen-binding portion thereof, with an improvedactivity and at least one retained property or characteristic, relativeto the parent antibody, or antigen-binding portion thereof, is obtained.

In a preferred embodiment, the contact positions are selected from thegroup consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52,L53, L55, L91, L92, L93, L94 and L96 and the other characteristic isselected from 1) preservation of non-crossreactivity with other proteinsor human tissues, 2) preservation of epitope recognition, i.e.recognizing p40 epitope preferably in the context of the p70 p40/p35heterodimer preventing binding interference from free, soluble p40and/or 3) to produce an antibody with a close to germline immunoglobulinsequence.

In another preferred embodiment, the hypermutation positions areselected from the group consisting of H30, H31, H31B, H32, H52, H56,H58, L30, L31, L32, L53 and L93 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence.

In a more preferred embodiment the residues for selective mutagenesisare selected from the preferred selective mutagenesis positions from thegroup consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,L32, L50, L91, L92, L93, L94 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence.

In a more preferred embodiment, the contact positions are selected fromthe group consisting of L50 and L94 and the other characteristic isselected from 1) preservation of non-crossreactivity with other proteinsor human tissues, 2) preservation of epitope recognition, i.e.recognizing p40 epitope preferably in the context of the p70 p40/p35heterodimer preventing binding interference from free, soluble p40and/or 3) to produce an antibody with a close to germline immunoglobulinsequence.

In another embodiment, the invention provides a method for improving theactivity of an antibody, or antigen-binding portion thereof, comprising:

a) providing a recombinant parent antibody or antigen-binding portionthereof; that was obtained by selection in a phage-display system butwhose activity cannot be further improved by mutagenesis in saidphage-display system;

b) selecting a preferred selective mutagenesis position, contact orhypermutation position within a complementarity determining region (CDR)for mutation, thereby identifying a selected contact or hypermutationposition;

c) individually mutating said selected preferred selective mutagenesispositions, contact or hypermutation position to at least two other aminoacid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof, and expressing said panel in anon-phage display system;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof for at least one other property or characteristic,wherein the property or characteristic is one that needs to be retained,until an antibody, or antigen-binding portion thereof, with an improvedactivity and at least one retained characteristic, relative to theparent antibody, or antigen-binding portion thereof, is obtained.

f) optionally, repeating steps a) through e) for at least one otherpreferred selective mutagenesis position, contact or hypermutationposition;

g) combining, in the parent antibody, or antigen-binding portionthereof, at least two individual activity enhancing amino acid residuesshown to have improved activity and at least on retained othercharacteristic, to form combination antibodies, or antigen-bindingportions thereof; and

h) evaluating the activity of the combination antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof; until an antibody, or antigen-bindingportion thereof, with an improved activity and at least one retainedproperty or characteristic, relative to the parent antibody, orantigen-binding portion thereof, is obtained.

In a preferred embodiment, the contact positions are selected from thegroup consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52,L53, L55, L91, L92, L93, L94 and L96 and the other characteristic isselected from 1) preservation of non-crossreactivity with other proteinsor human tissues, 2) preservation of epitope recognition, i.e.recognizing p40 epitope preferably in the context of the p70 p40/p35heterodimer preventing binding interference from free, soluble p40and/or 3) to produce an antibody with a close to germline immunoglobulinsequence.

In another preferred embodiment, the hypermutation positions areselected from the group consisting of H30, H31, H31B, H32, H52, H56,H58, L30, L31, L32, L53 and L93 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence.

In a more preferred embodiment the residues for selective mutagenesisare selected from the preferred selective mutagenesis positions from thegroup consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,L32, L50, L91, L92, L93, L94 and the other characteristic is selectedfrom 1) preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence.

In a more preferred embodiment, the contact positions are selected fromthe group consisting of L50 and L94 and the other characteristic isselected from 1) preservation of non-crossreactivity with other proteinsor human tissues, 2) preservation of epitope recognition, i.e.recognizing p40 epitope preferably in the context of the p70 p40/p35heterodimer preventing binding interference from free, soluble p40and/or 3) to produce an antibody with a close to germline immunoglobulinsequence.

IV. Modifications of Other CDR Residues

Ultimately, all CDR residues in a given antibody-antigen pair identifiedby any means to be required as activity enhancing amino acid residuesand/or required directly or indirectly for binding to the antigen and/orfor retaining other desirable properties or characteristics of theantibody. Such CDR residues are referred to as “preferred selectivemutagenesis positions”. It should be noted that in specificcircumstances that preferred selective mutagenesis residues can beidentified also by other means including co-crystallization of antibodyand antigen and molecular modeling.

If the preferred attempts to identify activity enhancing amino acidsfocussing on the preferred selective mutagenesis positions, contact orhypermutation positions described above are exhausted, or if additionalimprovements are required, the remaining CDR residues may be modified asdescribed below. It should be understood that the antibody could alreadybe modified in any one or more contact or hypermutation positionsaccording to the embodiments discussed above but may require furtherimprovements. Therefore, in another embodiment, the invention provides amethod for improving the activity of an antibody, or antigen-bindingportion thereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50,H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

c) individually mutating said selected position e.g., to at least twoother amino acid residues to thereby create a mutated antibody or apanel of mutated antibodies, or antigen-binding portions thereof;

d) evaluating the activity of the mutated antibody or the panel ofmutated antibodies, or antigen-binding portions thereof, relative to theparent antibody or antigen-binding portion thereof thereby identifyingan activity enhancing amino acid residue;

e) evaluating the mutated antibody or the panel of mutated antibodies,or antigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof, for changes in at least one otherproperty or characteristic until an antibody, or antigen-binding portionthereof, with an improved activity, relative to the parent antibody, orantigen-binding portion thereof, is obtained.

Preferably, the other characteristic or property is selected from 1)preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence

If mutagenesis of a single residue is not sufficient other residues canbe included; therefore, in another embodiment, the invention provides amethod for improving the activity of an antibody, or antigen-bindingportion thereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50,H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

c) individually mutating said selected position to at least two otheramino acid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof, thereby identifying an activityenhancing amino acid residue;

e) repeating steps b) through d) for at least one other CDR positionwhich is neither the position selected under b) nor a position at H30,H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96,H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,L94 and L96;

f) combining, in the parent antibody, or antigen-binding portionthereof, at least two individual activity enhancing amino acid residuesshown to have improved activity, to form combination antibodies, orantigen-binding portions thereof; and

g) evaluating the activity of the combination antibodies, orantigen-binding portions thereof with two activity enhancing amino acidresidues, relative to the parent antibody or antigen-binding portionthereof until an antibody, or antigen-binding portion thereof, with animproved activity, relative to the parent antibody, or antigen-bindingportion thereof, is obtained.

If the preferred attempts to identify activity enhancing amino acidsfocussing on the contact or hypermutation positions described above areexhausted, or if additional improvements are required, and the antibodyin question can not further be optimized by mutagenesis and phagedisplay (or related ribosome display) methods the remaining CDR residuesmay be modified as described below. It should be understood that theantibody could already be modified in any one or more preferredselective mutagenesis position, contact or hypermutation positionsaccording to the embodiments discussed above but may require furtherimprovements.

Therefore, in another embodiment, the invention provides a method forimproving the activity of an antibody, or antigen-binding portionthereof, comprising:

a) providing a recombinant parent antibody or antigen-binding portionthereof; that was obtained by selection in a phage-display system butwhose activity cannot be further improved by mutagenesis in saidphage-display system;

b) selecting a selecting an amino acid residue within a complementaritydetermining region (CDR) for mutation other than H30, H31, H31B, H32,H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101,L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and;

c) individually mutating said selected contact or hypermutation positionto at least two other amino acid residues to thereby create a panel ofmutated antibodies, or antigen-binding portions thereof, and expressingsaid panel in a non-phage display system;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof, for changes in at least one other property orcharacteristic, until an antibody, or antigen-binding portion thereof,with an improved activity, relative to the parent antibody, orantigen-binding portion thereof, is obtained.

Preferably, the other characteristic or property is selected from 1)preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence.

If a single mutagenesis is not sufficient to increase the affinity ofthe antibody other residues may be included in the mutagenesis.Therefore, in another embodiment, the invention provides a method forimproving the activity of an antibody, or antigen-binding portionthereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof thatwas obtained by selection in a phage-display system but whose activitycannot be further improved by mutagenesis in said phage-display system;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50,H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

c) individually mutating said selected position to at least two otheramino acid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof and expression in a non-phage displaysystem;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) repeating steps b) through d) for at least one other position whichis neither the position selected under b) nor a position at H30, H31,H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94;

g) combining, in the parent antibody, or antigen-binding portionthereof, at least two individual activity enhancing amino acid residuesshown to have improved activity, to form combination antibodies, orantigen-binding portions thereof; and

h) evaluating the activity and other property or characteristic of thecombination antibodies, or antigen-binding portions thereof with twoactivity enhancing amino acid residues, relative to the parent antibodyor antigen-binding portion thereof; until an antibody, orantigen-binding portion thereof, with an improved activity, relative tothe parent antibody, or antigen-binding portion thereof, is obtained.

Preferably, the other characteristic or property is selected from 1)preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence

The preferred attempts to identify activity enhancing amino acidsfocussing on the preferred selective mutagenesis positions, contact orhypermutation positions described may be exhausted, or additionalimprovements may be required, and it is important to retain otherproperties or characteristics of the antibody.

Therefore, in another embodiment, the invention provides a method forimproving the activity of an antibody, or antigen-binding portionthereof, without affecting other characteristics, comprising:

a) providing a parent antibody or antigen-binding portion thereof;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50,H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

c) individually mutating said selected position to at least two otheramino acid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof, for changes in at least one other property orcharacteristic until an antibody, or antigen-binding portion thereof,with an improved activity and retained other property or characteristic,relative to the parent antibody, or antigen-binding portion thereof, isobtained.

Preferably, the other characteristic or property is selected from 1)preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence

If mutagenesis of a single residue is not sufficient other residues canbe included; therefore, in another embodiment, the invention provides amethod for improving the activity of an antibody, or antigen-bindingportion thereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50,H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

c) individually mutating said selected position to at least two otheramino acid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof, thereby identifying an activityenhancing amino acid residue;

e.) evaluating the panel of mutated antibodies or antigen-bindingportions thereof, relative to the parent antibody or antigen-portionthereof, for changes in at least one other characteristic or property;

e) repeating steps b) through e) for at least one other CDR positionwhich is neither the position selected under b) nor a position at H30,H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96,H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,L94 and L96;

f) combining, in the parent antibody, or antigen-binding portionthereof, at least two individual activity enhancing amino acid residuesshown to have improved activity and not affecting at least one otherproperty or characteristic, to form combination antibodies, orantigen-binding portions thereof; and

g) evaluating the activity and the retention of at least one otherproperty or characteristic of the combination antibodies, orantigen-binding portions thereof with two activity enhancing amino acidresidues, relative to the parent antibody or antigen-binding portionthereof until an antibody, or antigen-binding portion thereof, with animproved activity and at least one retained other property orcharacteristic, relative to the parent antibody, or antigen-bindingportion thereof, is obtained.

Mutagenesis of the preferred selective mutagenesis position, contact andhypermutation residues may not have increased the affinity of theantibody sufficiently, and mutagenesis and the phage display method (orrelated ribosome display method) may no longer be useful and at leastone other characteristic or property of the antibody should be retained.

Therefore, in another embodiment the invention provides a method toimprove the affinity of an antibody or antigen-binding portion thereof,comprising:

a) providing a parent antibody or antigen-binding portion thereof thatwas obtained by selection in a phage-display system but whose activitycannot be further improved by mutagenesis in said phage-display system;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50,H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

c) individually mutating said selected position to at least two otheramino acid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof and expression in a non-phage displaysystem;

d) evaluating the activity of the panel of mutated antibodies, orantigen-binding portions thereof, relative to the parent antibody orantigen-binding portion thereof thereby identifying an activityenhancing amino acid residue;

e) evaluating the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof, for changes in at least one other property orcharacteristic until an antibody, or antigen-binding portion thereof,with an improved activity, relative to the parent antibody, orantigen-binding portion thereof, is obtained.

Preferably, the other characteristic or property is selected from 1)preservation of non-crossreactivity with other proteins or humantissues, 2) preservation of epitope recognition, i.e. recognizing p40epitope preferably in the context of the p70 p40/p35 heterodimerpreventing binding interference from free, soluble p40 and/or 3) toproduce an antibody with a close to germline immunoglobulin sequence

If mutagenesis of a single residue is not sufficient other residues canbe included; therefore, in another embodiment, the invention provides amethod for improving the activity of an antibody, or antigen-bindingportion thereof, comprising:

a) providing a parent antibody or antigen-binding portion thereof thatwas obtained by selection in a phage-display system but whose activitycannot be further improved by mutagenesis in said phage-display system;

b) selecting an amino acid residue within a complementarity determiningregion (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50,H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

c) individually mutating said selected position to at least two otheramino acid residues to thereby create a panel of mutated antibodies, orantigen-binding portions thereof and expression in a non-phage displaysystem;

d) evaluating the activity and retention of at least one other propertyor characteristic of the panel of mutated antibodies, or antigen-bindingportions thereof, relative to the parent antibody or antigen-bindingportion thereof, thereby identifying an activity enhancing amino acidresidue;

e) repeating steps b) through d) for at least one other CDR positionwhich is neither the position selected under b) nor a position at H30,H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96,H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,L94 and L96;

f) combining, in the parent antibody, or antigen-binding portionthereof, at least two individual activity enhancing amino acid residuesshown to have improved activity and not to affect at least one otherproperty or characteristic, to form combination antibodies, orantigen-binding portions thereof; and

g) evaluating the activity and retention of at least one property orcharacteristic of the combination antibodies, or antigen-bindingportions thereof with two activity enhancing amino acid residues,relative to the parent antibody or antigen-binding portion thereof untilan antibody, or antigen-binding portion thereof, with an improvedactivity and at least one other retained characteristic or property,relative to the parent antibody, or antigen-binding portion thereof, isobtained.

V. Expression of Antibodies

An antibody, or antibody portion, of the invention can be prepared byrecombinant expression of immunoglobulin light and heavy chain genes ina host cell. To express an antibody recombinantly, a host cell istransfected with one or more recombinant expression vectors carrying DNAfragments encoding the immunoglobulin light and heavy chains of theantibody such that the light and heavy chains are expressed in the hostcell and, preferably, secreted into the medium in which the host cellsare cultured, from which medium the antibodies can be recovered.Standard recombinant DNA methodologies are used to obtain antibody heavyand light chain genes, incorporate these genes into recombinantexpression vectors and introduce the vectors into host cells, such asthose described in Sambrook, Fritsch and Maniatis (eds), MolecularCloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,(1989), Ausubel, F. M. et al. (eds.) Current Protocols in MolecularBiology, Greene Publishing Associates, (1989) and in U.S. Pat. No.4,816,397 by Boss et al.

To obtain a DNA fragment encoding the heavy chain variable region of Joe9 wt or a Joe 9 wt-related antibody, antibodies specific for human IL-12were screened from human libraries and mutated, as described in sectionII. Once DNA fragments encoding Joe 9 wt or Joe 9 wt-related VH and VLsegments are obtained, mutagenesis of these sequences is carried out bystandard methods, such as PCR site directed mutagenesis (PCR-mediatedmutagenesis in which the mutated nucleotides are incorporated into thePCR primers such that the PCR product contains the mutations) or othersite-directed mutagenesis methods. Human IL-12 antibodies that displayeda level of activity and binding specificity/affinity that was desirable,for example J695, were further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a VL- or VH-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region and any allotypicvariant therein as described in Kabat, (Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242),but most preferably is an IgG1 or IgG4 constant region. For a Fabfragment heavy chain gene, the VH-encoding DNA can be operatively linkedto another DNA molecule encoding only the heavy chain CH1 constantregion.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but most preferably is alambda constant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

To express the antibodies, or antibody portions of the invention, DNAsencoding partial or full-length light and heavy chains, obtained asdescribed above, are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalcontrol sequences. In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vector or, more typically, both genesare inserted into the same expression vector. The antibody genes areinserted into the expression vector by standard methods (e.g., ligationof complementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present).Prior to insertion of the J695 or J695-related light or heavy chainsequences, the expression vector may already carry antibody constantregion sequences. For example, one approach to converting the J695 orJ695-related VH and VL sequences to full-length antibody genes is toinsert them into expression vectors already encoding heavy chainconstant and light chain constant regions, respectively, such that theVH segment is operatively linked to the CH segment(s) within the vectorand the VL segment is operatively linked to the CL segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the antibodychain from a host cell. The antibody chain gene can be cloned into thevector such that the signal peptide is linked in-frame to the aminoterminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, seee.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al., U.S. Pat.No. 5,464,758 by Bujard et al. and U.S. Pat. No. 5,654,168 by Bujard etal.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr⁻ host cellswith methotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Preferred mammalianhost cells for expressing the recombinant antibodies of the inventioninclude Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells,described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA77:4216-4220, used with a DHFR selectable marker, e.g., as described inR. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSOmyeloma cells, COS cells and SP2 cells. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods.

Host cells can also be used to produce portions of intact antibodies,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding either the light chain or the heavy chain (but notboth) of an antibody of this invention. Recombinant DNA technology mayalso be used to remove some or all of the DNA encoding either or both ofthe light and heavy chains that is not necessary for binding to hIL-12The molecules expressed from such truncated DNA molecules are alsoencompassed by the antibodies of the invention. In addition,bifunctional antibodies may be produced in which one heavy and one lightchain are an antibody of the invention and the other heavy and lightchain are specific for an antigen other than hIL-12 by crosslinking anantibody of the invention to a second antibody by standard chemicalcrosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to enhancer/promoter regulatory elements (e.g., derived fromSV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLPpromoter regulatory element or an SV40 enhancer/AdMLP promoterregulatory element) to drive high levels of transcription of the genes.The recombinant expression vector also carries a DHFR gene, which allowsfor selection of CHO cells that have been transfected with the vectorusing methotrexate selection/amplification. The selected transformanthost cells are culture to allow for expression of the antibody heavy andlight chains and intact antibody is recovered from the culture medium.Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the antibody from theculture medium. Antibodies or antigen-binding portions thereof of theinvention can be expressed in an animal (e.g., a mouse) that istransgenic for human immunoglobulin genes (see e.g., Taylor, L. D. etal. (1992) Nucl. Acids Res. 20: 6287-6295). Plant cells can also bemodified to create transgenic plants that express the antibody orantigen binding portion thereof, of the invention.

In view of the foregoing, another aspect of the invention pertains tonucleic acid, vector and host cell compositions that can be used forrecombinant expression of the antibodies and antibody portions of theinvention. Preferably, the invention features isolated nucleic acidsthat encode CDRs of J695, or the full heavy and/or light chain variableregion of J695. Accordingly, in one embodiment, the invention featuresan isolated nucleic acid encoding an antibody heavy chain variableregion that encodes the J695 heavy chain CDR3 comprising the amino acidsequence of SEQ ID NO: 25. Preferably, the nucleic acid encoding theantibody heavy chain variable region further encodes a J695 heavy chainCDR2 which comprises the amino acid sequence of SEQ ID NO: 27. Morepreferably, the nucleic acid encoding the antibody heavy chain variableregion further encodes a J695 heavy chain CDR1 which comprises the aminoacid sequence of SEQ ID NO: 29. Even more preferably, the isolatednucleic acid encodes an antibody heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 31 (the full VH region of J695).

In other embodiments, the invention features an isolated nucleic acidencoding an antibody light chain variable region that encodes the J695light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26.Preferably, the nucleic acid encoding the antibody light chain variableregion further encodes a J695 light chain CDR2 which comprises the aminoacid sequence of SEQ ID NO: 28. More preferably, the nucleic acidencoding the antibody light chain variable region further encodes a J695light chain CDR1 which comprises the amino acid sequence of SEQ ID NO:30. Even more preferably, the isolated nucleic acid encodes an antibodylight chain variable region comprising the amino acid sequence of SEQ IDNO: 32 (the full VL region of J695).

The invention also provides recombinant expression vectors encoding bothan antibody heavy chain and an antibody light chain. For example, in oneembodiment, the invention provides a recombinant expression vectorencoding:

-   -   a) an antibody heavy chain having a variable region comprising        the amino acid sequence of SEQ ID NO: 31; and    -   b) an antibody light chain having a variable region comprising        the amino acid sequence of SEQ ID NO: 32.

The invention also provides host cells into which one or more of therecombinant expression vectors of the invention have been introduced.Preferably, the host cell is a mammalian host cell, more preferably thehost cell is a CHO cell, an NSO cell or a COS cell. Still further theinvention provides a method of synthesizing a recombinant human antibodyof the invention by culturing a host cell of the invention in a suitableculture medium until a recombinant human antibody of the invention issynthesized. The method can further comprise isolating the recombinanthuman antibody from the culture medium.

VI. Pharmaceutical Compositions and Pharmaceutical Administration

The antibodies and antibody-portions of the invention can beincorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises an antibody or antibody portion of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody or antibody portion.

The antibodies and antibody-portions of the invention can beincorporated into a pharmaceutical composition suitable for parenteraladministration. Preferably, the antibody or antibody-portions will beprepared as an injectable solution containing 0.1-250 mg/ml antibody.The injectable solution can be composed of either a liquid orlyophilized dosage form in a flint or amber vial, ampule or pre-filledsyringe. The buffer can be L-histidine (1-50 mM), optimally 5-10 mM, atpH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but arenot limited to, sodium succinate, sodium citrate, sodium phosphate orpotassium phosphate. Sodium chloride can be used to modify the toxicityof the solution at a concentration of 0-300 mM (optimally 150 mM for aliquid dosage form). Cryoprotectants can be included for a lyophilizeddosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Othersuitable cryoprotectants include trehalose and lactose. Bulking agentscan be included for a lyophilized dosage form, principally 1-10%mannitol (optimally 2-4%). Stabilizers can be used in both liquid andlyophilized dosage forms, principally 1-50 mM L-Methionine (optimally5-10 mM). Other suitable bulking agents include glycine, arginine, canbe included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%).Additional surfactants include but are not limited to polysorbate 20 andBRIJ surfactants.

In a preferred embodiment, the pharmaceutical composition includes theantibody at a dosage of about 0.01 mg/kg-10 mg/kg. More preferreddosages of the antibody include 1 mg/kg administered every other week,or 0.3 mg/kg administered weekly.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, the antibody is administered by intravenous infusion orinjection. In another preferred embodiment, the antibody is administeredby intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

The antibodies and antibody-portions of the present invention can beadministered by a variety of methods known in the art, although for manytherapeutic applications, the preferred route/mode of administration issubcutaneous injection, intravenous injection or infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

In certain embodiments, an antibody or antibody portion of the inventionmay be orally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion ofthe invention is coformulated with and/or coadministered with one ormore additional therapeutic agents that are useful for treatingdisorders in which IL-12 activity is detrimental. For example, ananti-hIL-12 antibody or antibody portion of the invention may becoformulated and/or coadministered with one or more additionalantibodies that bind other targets (e.g., antibodies that bind othercytokines or that bind cell surface molecules). Furthermore, one or moreantibodies of the invention may be used in combination with two or moreof the foregoing therapeutic agents. Such combination therapies mayadvantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies. It will be appreciated by the skilledpractitioner that when the antibodies of the invention are used as partof a combination therapy, a lower dosage of antibody may be desirablethan when the antibody alone is administered to a subject (e.g., asynergistic therapeutic effect may be achieved through the use ofcombination therapy which, in turn, permits use of a lower dose of theantibody to achieve the desired therapeutic effect).

Interleukin 12 plays a critical role in the pathology associated with avariety of diseases involving immune and inflammatory elements. Thesediseases include, but are not limited to, rheumatoid arthritis,osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriaticarthritis, reactive arthritis, spondyloarthropathy, systemic lupuserythematosus, Crohn's disease, ulcerative colitis, inflammatory boweldisease, insulin dependent diabetes mellitus, thyroiditis, asthma,allergic diseases, psoriasis, dermatitis scleroderma, atopic dermatitis,graft versus host disease, organ transplant rejection, acute or chronicimmune disease associated with organ transplantation, sarcoidosis,atherosclerosis, disseminated intravascular coagulation, Kawasaki'sdisease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome,Wegener's granulomatosis, Henoch-Schoenlein purpurea, microscopicvasculitis of the kidneys, chronic active hepatitis, uveitis, septicshock, toxic shock syndrome, sepsis syndrome, cachexia, infectiousdiseases, parasitic diseases, acquired immunodeficiency syndrome, acutetransverse myelitis, Huntington's chorea, Parkinson's disease,Alzheimer's disease, stroke, primary biliary cirrhosis, hemolyticanemia, malignancies, heart failure, myocardial infarction, Addison'sdisease, sporadic, polyglandular deficiency type I and polyglandulardeficiency type II, Schmidt's syndrome, adult (acute) respiratorydistress syndrome, alopecia, alopecia areata, seronegative arthopathy,arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative coliticarthropathy, enteropathic synovitis, chlamydia, yersinia and salmonellaassociated arthropathy, spondyloarthopathy, atheromatousdisease/arteriosclerosis, atopic allergy, autoimmune bullous disease,pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease,autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia,acquired pernicious anaemia, juvenile pernicious anaemia, myalgicencephalitis/Royal Free Disease, chronic mucocutaneous candidiasis,giant cell arteritis, primary sclerosing hepatitis, cryptogenicautoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome,Acquired Immunodeficiency Related Diseases, Hepatitis C, common variedimmunodeficiency (common variable hypogammaglobulinaemia), dilatedcardiomyopathy, female infertility, ovarian failure, premature ovarianfailure, fibrotic lung disease, cryptogenic fibrosing alveolitis,post-inflammatory interstitial lung disease, interstitial pneumonitis,connective tissue disease associated interstitial lung disease, mixedconnective tissue disease associated lung disease, systemic sclerosisassociated interstitial lung disease, rheumatoid arthritis associatedinterstitial lung disease, systemic lupus erythematosus associated lungdisease, dermatomyositis/polymyositis associated lung disease, Sjögren'sdisease associated lung disease, ankylosing spondylitis associated lungdisease, vasculitic diffuse lung disease, haemosiderosis associated lungdisease, drug-induced interstitial lung disease, radiation fibrosis,bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocyticinfiltrative lung disease, postinfectious interstitial lung disease,gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis(classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis(anti-LKM antibody hepatitis), autoimmune mediated hypoglycemia, type Binsulin resistance with acanthosis nigricans, hypoparathyroidism, acuteimmune disease associated with organ transplantation, chronic immunedisease associated with organ transplantation, osteoarthrosis, primarysclerosing cholangitis, idiopathic leucopenia, autoimmune neutropenia,renal disease NOS, glomerulonephritides, microscopic vasulitis of thekidneys, lyme disease, discoid lupus erythematosus, male infertilityidiopathic or NOS, sperm autoimmunity, multiple sclerosis (allsubtypes), insulin-dependent diabetes mellitus, sympathetic ophthalmia,pulmonary hypertension secondary to connective tissue disease,Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa,acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemicsclerosis, Takayasu's disease/arteritis, autoimmune thrombocytopenia,idiopathic thrombocytopenia, autoimmune thyroid disease,hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto'sdisease), atrophic autoimmune hypothyroidism, primary myxoedema,phacogenic uveitis, primary vasculitis and vitiligo. The humanantibodies, and antibody portions of the invention can be used to treatautoimmune diseases, in particular those associated with inflammation,including, rheumatoid spondylitis, allergy, autoimmune diabetes,autoimmune uveitis.

Preferably, the antibodies of the invention or antigen-binding portionsthereof, are used to treat rheumatoid arthritis, Crohn's disease,multiple sclerosis, insulin dependent diabetes mellitus and psoriasis,as described in more detail in section VII.

A human antibody, or antibody portion, of the invention also can beadministered with one or more additional therapeutic agents useful inthe treatment of autoimmune and inflammatory diseases.

Antibodies of the invention, or antigen binding portions thereof can beused alone or in combination to treat such diseases. It should beunderstood that the antibodies of the invention or antigen bindingportion thereof can be used alone or in combination with an additionalagent, e.g., a therapeutic agent, said additional agent being selectedby the skilled artisan for its intended purpose. For example, theadditional agent can be a therapeutic agent art-recognized as beinguseful to treat the disease or condition being treated by the antibodyof the present invention. The additional agent also can be an agentwhich imparts a beneficial attribute to the therapeutic compositione.g., an agent which effects the viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth below are illustrative forpurposes and not intended to be limited. The combinations which are partof this invention can be the antibodies of the present invention and atleast one additional agent selected from the lists below. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

Preferred combinations are non-steroidal anti-inflammatory drug(s) alsoreferred to as NSAIDS which include drugs like ibuprofen. Otherpreferred combinations are corticosteroids including prednisolone; thewell known side-effects of steroid use can be reduced or even eliminatedby tapering the steroid dose required when treating patients incombination with the anti-IL-12 antibodies of this invention.Non-limiting examples of therapeutic agents for rheumatoid arthritiswith which an antibody, or antibody portion, of the invention can becombined include the following: cytokine suppressive anti-inflammatorydrug(s) (CSAIDs); antibodies to or antagonists of other human cytokinesor growth factors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8,IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of theinvention, or antigen binding portions thereof, can be combined withantibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25,CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, or theirligands including CD154 (gp39 or CD40L).

Preferred combinations of therapeutic agents may interfere at differentpoints in the autoimmune and subsequent inflammatory cascade; preferredexamples include TNF antagonists like chimeric, humanized or human TNFantibodies, D2E7, (U.S. application Ser. No. 08/599,226 filed Feb. 9,1996), cA2 (Remicadem), CDP 571, anti-TNF antibody fragments (e.g.,CDP870), and soluble p55 or p75 TNF receptors, derivatives thereof,(p75TNFR1gG (Enbrel™) or p55TNFR1gG (Lenercept), soluble IL-13 receptor(sIL-13), and also TNFα converting enzyme (TACE) inhibitors; similarlyIL-1 inhibitors (e.g., Interleukin-1-converting enzyme inhibitors, suchas Vx740, or IL-1RA etc.) may be effective for the same reason. Otherpreferred combinations include Interleukin 11, anti-P7s and p-selectinglycoprotein ligand (PSGL). Yet another preferred combination are otherkey players of the autoimmune response which may act parallel to,dependent on or in concert with IL-12 function; especially preferred areIL-18 antagonists including IL-18 antibodies or soluble IL-18 receptors,or IL-18 binding proteins. It has been shown that IL-12 and IL-18 haveoverlapping but distinct functions and a combination of antagonists toboth may be most effective. Yet another preferred combination arenon-depleting anti-CD4 inhibitors. Yet other preferred combinationsinclude antagonists of the co-stimulatory pathway CD80 (B7.1) or CD86(B7.2) including antibodies, soluble receptors or antagonistic ligands.

The antibodies of the invention, or antigen binding portions thereof,may also be combined with agents, such as methotrexate, 6-MP,azathioprine sulphasalazine, mesalazine, olsalazinechloroquinine/hydroxychloroquine, pencillamine, aurothiomalate(intramuscular and oral), azathioprine, cochicine, corticosteroids(oral, inhaled and local injection), beta-2 adrenoreceptor agonists(salbutamol, terbutaline, salmeteral), xanthines (theophylline,aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium andoxitropium, cyclosporin, FK506, rapamycin, mycophenolate mofetil,leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such asprednisolone, phosphodiesterase inhibitors, adensosine agonists,antithrombotic agents, complement inhibitors, adrenergic agents, agentswhich interfere with signalling by proinflammatory cytokines such asTNFα or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1βconverting enzyme inhibitors (e.g., Vx740), anti-P7s, p-selectinglycoprotein ligand (PSGL), TNFα converting enzyme (TACE) inhibitors,T-cell signalling inhibitors such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNFreceptors and the derivatives p75TNFRIgG (Enbrel™) and p55TNFRIgG(Lenercept), sIL-1RI, sIL-1RII, sIL-6R, soluble IL-13 receptor (sIL-13))and antiinflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13 andTGFβ). Preferred combinations include methotrexate or leflunomide and inmoderate or severe rheumatoid arthritis cases, cyclosporine.

Non-limiting examples of therapeutic agents for inflammatory boweldisease with which an antibody, or antibody portion, of the inventioncan be combined include the following: budenoside; epidermal growthfactor; corticosteroids; cyclosporin, sulfasalazine; aminosalicylates;6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors;mesalamine; olsalazine; balsalazide; antioxidants; thromboxaneinhibitors; IL-1 receptor antagonists; anti-IL-1β monoclonal antibodies;anti-IL-6 monoclonal antibodies; growth factors; elastase inhibitors;pyridinyl-imidazole compounds; antibodies to or antagonists of otherhuman cytokines or growth factors, for example, TNF, LT, IL-1, IL-2,IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF.Antibodies of the invention, or antigen binding portions thereof, can becombined with antibodies to cell surface molecules such as CD2, CD3,CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands. Theantibodies of the invention, or antigen binding portions thereof, mayalso be combined with agents, such as methotrexate, cyclosporin, FK506,rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example,ibuprofen, corticosteroids such as prednisolone, phosphodiesteraseinhibitors, adenosine agonists, antithrombotic agents, complementinhibitors, adrenergic agents, agents which interfere with signalling byproinflammatory cytokines such as TNFα or IL-1 (e.g. IRAK, NIK, IKK, p38or MAP kinase inhibitors), IL-1β converting enzyme inhibitors (e.g.,Vx740), anti-P7s, p-selectin glycoprotein ligand (PSGL), TNFα convertingenzyme inhibitors, T-cell signalling inhibitors such as kinaseinhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNFreceptors, sIL-1RI, sIL-1RII, sIL-6R, soluble IL-13 receptor (sIL-13))and antiinflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13 andTGFβ).

Preferred examples of therapeutic agents for Crohn's disease in which anantibody or an antigen binding portion can be combined include thefollowing: TNF antagonists, for example, anti-TNF antibodies, D2E7 (U.S.application Ser. No. 08/599,226, filed Feb. 9, 1996), cA2 (Remicade™),CDP 571, anti-TNF antibody fragments (e.g., CDP870), TNFR-Ig constructs(p75TNFRIgG (Enbrel™) and p55TNFRIgG (Lenercept)), anti-P7s, p-selectinglycoprotein ligand (PSGL), soluble IL-13 receptor (sIL-13), and PDE4inhibitors. Antibodies of the invention or antigen binding portionsthereof, can be combined with corticosteroids, for example, budenosideand dexamethasone. Antibodies of the invention or antigen bindingportions thereof, may also be combined with agents such assulfasalazine, 5-aminosalicylic acid and olsalazine, and agents whichinterfere with synthesis or action of proinflammatory cytokines such asIL-1, for example, IL-1β converting enzyme inhibitors (e.g., Vx740) andIL-1ra. Antibodies of the invention or antigen binding portion thereofmay also be used with T cell signaling inhibitors, for example, tyrosinekinase inhibitors 6-mercaptopurines. Antibodies of the invention orantigen binding portions thereof, can be combined with IL-11.

Non-limiting examples of therapeutic agents for multiple sclerosis withwhich an antibody, or antibody portion, of the invention can be combinedinclude the following: corticosteroids; prednisolone;methylprednisolone; azathioprine; cyclophosphamide; cyclosporine;methotrexate; 4-aminopyridine; tizanidine; interferon-β1a (Avonex;Biogen); interferon-β1b (Betaseron; Chiron/Berlex); Copolymer 1 (Cop-1;Copaxone; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen;intravenous immunoglobulin; clabribine; antibodies to or antagonists ofother human cytokines or growth factors, for example, TNF, LT, IL-1,IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, andPDGF. Antibodies of the invention, or antigen binding portions thereof,can be combined with antibodies to cell surface molecules such as CD2,CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 ortheir ligands. The antibodies of the invention, or antigen bindingportions thereof, may also be combined with agents, such asmethotrexate, cyclosporine, FK506, rapamycin, mycophenolate mofetil,leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such asprednisolone, phosphodiesterase inhibitors, adensosine agonists,antithrombotic agents, complement inhibitors, adrenergic agents, agentswhich interfere with signalling by proinflammatory cytokines such asTNFα or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1βconverting enzyme inhibitors (e.g., Vx740), anti-P7s, p-selectinglycoprotein ligand (PSGL), TACE inhibitors, T-cell signallinginhibitors such as kinase inhibitors, metalloproteinase inhibitors,sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin convertingenzyme inhibitors, soluble cytokine receptors and derivatives thereof(e.g. soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R,soluble IL-13 receptor (sIL-13)) and antiinflammatory cytokines (e.g.IL-4, IL-10, IL-13 and TGFβ).

Preferred examples of therapeutic agents for multiple sclerosis in whichthe antibody or antigen binding portion thereof can be combined toinclude interferon-β, for example, IFNβ1a and IFNβ1b; copaxone,corticosteroids, IL-1 inhibitors, TNF inhibitors, and antibodies to CD40ligand and CD80.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody portion may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody or antibody portion to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 0.01-20 mg/kg, more preferably 1-10 mg/kg, even morepreferably 0.3-1 mg/kg. It is to be noted that dosage values may varywith the type and severity of the condition to be alleviated. It is tobe further understood that for any particular subject, specific dosageregimens should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed composition.

VII. Uses of the Antibodies of the Invention

Given their ability to bind to hIL-12, the anti-hIL-12 antibodies, orportions thereof, of the invention can be used to detect hIL-12 (e.g.,in a biological sample, such as serum or plasma), using a conventionalimmunoassay, such as an enzyme linked immunosorbent assays (ELISA), anradioimmunoassay (RIA) or tissue immunohistochemistry. The inventionprovides a method for detecting hIL-12 in a biological sample comprisingcontacting a biological sample with an antibody, or antibody portion, ofthe invention and detecting either the antibody (or antibody portion)bound to hIL-12 or unbound antibody (or antibody portion), to therebydetect hIL-12 in the biological sample. The antibody is directly orindirectly labeled with a detectable substance to facilitate detectionof the bound or unbound antibody. Suitable detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ¹²⁵I,¹³¹I, ³⁵S or ³H.

Alternative to labeling the antibody, hIL-12 can be assayed inbiological fluids by a competition immunoassay utilizing rhIL-12standards labeled with a detectable substance and an unlabeledanti-hIL-12 antibody. In this assay, the biological sample, the labeledrhIL-12 standards and the anti-hIL-12 antibody are combined and theamount of labeled rhIL-12 standard bound to the unlabeled antibody isdetermined. The amount of hIL-12 in the biological sample is inverselyproportional to the amount of labeled rhIL-12 standard bound to theanti-hIL-12 antibody.

The Y61 and J695 antibodies of the invention can also be used to detectIL-12 from species other than humans, in particular IL-12 from primates.For example, Y61 can be used to detect IL-12 in the cynomolgus monkeyand the rhesus monkey. J695 can be used to detect IL-12 in thecynomolgus monkey, rhesus monkey, and baboon. However, neither antibodycross reacts with mouse or rat IL-12 (see Example 3, subsection F).

The antibodies and antibody portions of the invention are capable ofneutralizing hIL-12 activity in vitro (see Example 3) and in vivo (seeExample 4). Accordingly, the antibodies and antibody portions of theinvention can be used to inhibit IL-12 activity, e.g., in a cell culturecontaining hIL-12, in human subjects or in other mammalian subjectshaving IL-12 with which an antibody of the invention cross-reacts (e.g.primates such as baboon, cynomolgus and rhesus). In a preferredembodiment, the invention provides an isolated human antibody, orantigen-binding portion thereof, that neutralizes the activity of humanIL-12, and at least one additional primate IL-12 selected from the groupconsisting of baboon IL-12, marmoset IL-12, chimpanzee IL-12, cynomolgusIL-12 and rhesus IL-12, but which does not neutralize the activity ofthe mouse IL-12. Preferably, the IL-12 is human IL-12. For example, in acell culture containing, or suspected of containing hIL-12, an antibodyor antibody portion of the invention can be added to the culture mediumto inhibit hIL-12 activity in the culture.

In another embodiment, the invention provides a method for inhibitingIL-12 activity in a subject suffering from a disorder in which IL-12activity is detrimental. IL-12 has been implicated in thepathophysiology of a wide variety of disorders (Windhagen et al., (1995)J. Exp. Med. 182: 1985-1996; Morita et al. (1998) Arthritis andRheumatism. 41: 306-314; Bucht et al., (1996) Clin. Exp. Immunol. 103:347-367; Fais et al. (1994) J. Interferon Res. 14:235-238; Parronchi etal., (1997) Am. J. Path. 150:823-832; Monteleone et al., (1997)Gastroenterology. 112:1169-1178, and Berrebi et al., (1998) Am. J. Path152:667-672; Parronchi et al (1997) Am. J. Path. 150:823-832). Theinvention provides methods for inhibiting IL-12 activity in a subjectsuffering from such a disorder, which method comprises administering tothe subject an antibody or antibody portion of the invention such thatIL-12 activity in the subject is inhibited. Preferably, the IL-12 ishuman IL-12 and the subject is a human subject. Alternatively, thesubject can be a mammal expressing a IL-12 with which an antibody of theinvention cross-reacts. Still further the subject can be a mammal intowhich has been introduced hIL-12 (e.g., by administration of hIL-12 orby expression of an hIL-12 transgene). An antibody of the invention canbe administered to a human subject for therapeutic purposes (discussedfurther below). Moreover, an antibody of the invention can beadministered to a non-human mammal expressing a IL-12 with which theantibody cross-reacts for veterinary purposes or as an animal model ofhuman disease. Regarding the latter, such animal models may be usefulfor evaluating the therapeutic efficacy of antibodies of the invention(e.g., testing of dosages and time courses of administration).

As used herein, the phrase “a disorder in which IL-12 activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of IL-12 in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which IL-12activity is detrimental is a disorder in which inhibition of IL-12activity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of IL-12 in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofIL-12 in serum, plasma, synovial fluid, etc. of the subject), which canbe detected, for example, using an anti-IL-12 antibody as describedabove. There are numerous examples of disorders in which IL-12 activityis detrimental. In one embodiment, the antibodies or antigen bindingportions thereof, can be used in therapy to treat the diseases ordisorders described herein. In another embodiment, the antibodies orantigen binding portions thereof, can be used for the manufacture of amedicine for treating the diseases or disorders described herein. Theuse of the antibodies and antibody portions of the invention in thetreatment of a few non-limiting specific disorders is discussed furtherbelow:

A. Rheumatoid Arthritis:

Interleukin-12 has been implicated in playing a role in inflammatorydiseases such as rheumatoid arthritis. Inducible IL-12p40 message hasbeen detected in synovia from rheumatoid arthritis patients and IL-12has been shown to be present in the synovial fluids from patients withrheumatoid arthritis (see e.g., Morita et al., (1998) Arthritis andRheumatism 41: 306-314). IL-12 positive cells have been found to bepresent in the sublining layer of the rheumatoid arthritis synovium. Thehuman antibodies, and antibody portions of the invention can be used totreat, for example, rheumatoid arthritis, juvenile rheumatoid arthritis,Lyme arthritis, rheumatoid spondylitis, osteoarthritis and goutyarthritis. Typically, the antibody, or antibody portion, is administeredsystemically, although for certain disorders, local administration ofthe antibody or antibody portion may be beneficial. An antibody, orantibody portion, of the invention also can be administered with one ormore additional therapeutic agents useful in the treatment of autoimmunediseases.

In the collagen induced arthritis (CIA) murine model for rheumatoidarthritis, treatment of mice with an anti-IL-12 mAb (rat anti-mouseIL-12 monoclonal antibody, C17.15) prior to arthritis profoundlysuppressed the onset, and reduced the incidence and severity of disease.Treatment with the anti-IL-12 mAb early after onset of arthritis reducedseverity, but later treatment of the mice with the anti-IL-12 mAb afterthe onset of disease had minimal effect on disease severity.

B. Crohn's Disease

Interleukin-12 also plays a role in the inflammatory bowel disease,Crohn's disease. Increased expression of IFN-γ and IL-12 occurs in theintestinal mucosa of patients with Crohn's disease (see e.g., Fais etal., (1994) J. Interferon Res. 14: 235-238; Parronchi et al., (1997)Amer. J. Pathol. 150: 823-832; Monteleone et al., (1997)Gastroenterology 112: 1169-1178; Berrebi et al., (1998) Amer. J. Pathol.152: 667-672). Anti-IL-12 antibodies have been shown to suppress diseasein mouse models of colitis, e.g., TNBS induced colitis IL-2 knockoutmice, and recently in IL-10 knock-out mice. Accordingly, the antibodies,and antibody portions, of the invention, can be used in the treatment ofinflammatory bowel diseases.

C. Multiple Sclerosis

Interleukin-12 has been implicated as a key mediator of multiplesclerosis. Expression of the inducible IL-12 p40 message or IL-12 itselfcan be demonstrated in lesions of patients with multiple sclerosis(Windhagen et al., (1995) J. Exp. Med. 182: 1985-1996, Drulovic et al.,(1997) J. Neurol. Sci. 147: 145-150). Chronic progressive patients withmultiple sclerosis have elevated circulating levels of IL-12.Investigations with T-cells and antigen presenting cells (APCs) frompatients with multiple sclerosis revealed a self-perpetuating series ofimmune interactions as the basis of progressive multiple sclerosisleading to a Th1-type immune response. Increased secretion of IFN-γ fromthe T cells led to increased IL-12 production by APCs, which perpetuatedthe cycle leading to a chronic state of a Th1-type immune activation anddisease (Balashov et al., (1997) Proc. Natl. Acad. Sci. 94: 599-603).The role of IL-12 in multiple sclerosis has been investigated usingmouse and rat experimental allergic encephalomyelitis (EAE) models ofmultiple sclerosis. In a relapsing-remitting EAE model of multiplesclerosis in mice, pretreatment with anti-IL-12 mAb delayed paralysisand reduced clinical scores. Treatment with anti-IL-12 mAb at the peakof paralysis or during the subsequent remission period reduced clinicalscores. Accordingly, the antibodies or antigen binding portions thereofof the invention may serve to alleviate symptoms associated withmultiple sclerosis in humans.

D. Insulin-Dependent Diabetes Mellitus

Interleukin-12 has been implicated as an important mediator ofinsulin-dependent diabetes mellitus (IDDM). IDDM was induced in NOD miceby administration of IL-12, and anti-IL-12 antibodies were protective inan adoptive transfer model of IDDM. Early onset IDDM patients oftenexperience a so-called “honeymoon period” during which some residualislet cell function is maintained. These residual islet cells produceinsulin and regulate blood glucose levels better than administeredinsulin. Treatment of these early onset patients with an anti-IL-12antibody may prevent further destruction of islet cells, therebymaintaining an endogenous source of insulin.

E. Psoriasis

Interleukin-12 has been implicated as a key mediator in psoriasis.Psoriasis involves acute and chronic skin lesions that are associatedwith a TH1-type cytokine expression profile. (Hamid et al. (1996) J.Allergy Clin. Immunol. 1:225-231; Turka et al. (1995) Mol. Med.1:690-699). IL-12 p35 and p40 mRNAs were detected in diseased human skinsamples. Accordingly, the antibodies or antigen binding portions thereofof the invention may serve to alleviate chronic skin disorders suchpsoriasis.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references, including literature references, issued patents,and published patent applications, as cited throughout this applicationare hereby expressly incorporated by reference. It should further beunderstood that the contents of all the tables attached hereto (seeAppendix A) are incorporated by reference.

TABLE 1 VH3 Family Germline Amino Acid SequenceNumbering according to Kabat (Joe9 VH included for cmmparison) SEQ germ-ID line NO: VH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 2223 24 25 26 594 dp-29 E V Q L V E S G G G L V Q P G G S L R L S C A A SG 595 DP-30 E V Q L V E S G G G L V Q P G G S L R L S C A A S G 596HC15-7 E V Q L V E S G G G L V Q P G G S L R L S C A A S G 597 VHD26 E VQ L L E S G G G L V Q P G G S L R L S C A A S G 598 DP-31 E V Q L V E SG G G L V Q P G R S L R L S C A A S G 599 DP-32 E V Q L V E S G G G V VR P G G S L R L S C A A S G 600 DP-33 E V Q L V E S G G V V V Q P G G SL R L S C A A S G 601 dp-35 Q V Q L V E S G G G L V K P G G S L R L S CA A S G 602 VH3-8 Q V Q L L E S G G G L V K P G G S L R L S C A A S G603 yac-9 E V Q L V E S G G G L V Q P G G S L K L S C A A S G 604 dp-38E V Q L V E S G G G L V K P G G S L R L S C A A S G 605 LSG2 E V Q L V ES G G G L V K P G G S L R L S C A A S G 606 LSG3 E V Q L V E S G G G L VK P G G S L R L S C A A S G 607 LSG4 E V Q L V E S G G G L V K P G G S LR L S C A A S G 608 LSG6 E V Q L V E S G G G L V K P G G S L R L S C A AS G 609 v3-15 E V Q L V E S G G A L V K P G G S L R L S C A A S G 610dp-39 E V Q L V E S G G G L V Q P G G S L R L S C P A S G 611 dp-40 E VQ L V E S G G G L V Q P G G S L R L S C A A S G 612 dp-59 E V Q L V E SG G G L V Q P G G S L R L S C A A S G 613 v3-16p E V Q L V E S G G G L VQ P G G S L R L S C A A S G 614 v3-19p T V Q L V E S G G G L V E P G G SL R L S C A A S G 615 v3-13 E V H L V E S G G G L V Q P G G A L R L S CA A S G 616 DP-42 E V Q L V E T G G G L I Q P G G S L R L S C A A S G617 dp-44 E V Q L V Q S G G G L V H P G G S L R L S C A G S G 618 DP-45E V Q L V Q S G G G L V Q P G G S L R L S C A G S G 619 dp-47 E V Q L LE S G G G L V Q P G G S L R L S C A A S G 620 flm E V Q L V E S G G G LV Q P G G S L R L S C S A S G 621 P1 E V Q L V E S G G G L V Q P G G S LR L S C S A S G 622 v3-64 E V Q L V E S G G G L V Q P G G S L R L S C AA S G 623 vh26 E V Q L L E S G G G L V Q P G G S L R L S C A A S G 624B25 Q V Q L V E S G G G V V Q P G R S L R L S C A A S G 625 b32e Q V Q LV E S G G G V V Q P G R S L R L S C A A S G 626 B37 Q V Q L V E S G G GV V Q P G R S L R L S C A A S G 627 B43 Q V Q L V E S G G G V V Q P G RS L R L S C A A S G 628 B48 Q V Q L V E S G G G V V Q P G R S L R L S CA A S G 629 B52 Q V Q L V E S G G G V V Q P G R S L R L S C A A S G 630B54 Q V Q L V E S G G G V V Q P G R S L R L S C A A S G 631 cos-B Q V QL V E S G G G V V Q P G R S L R L S C A A S G 632 dp-46 Q V Q L V E S GG G V V Q P G R S L R L S C A A S G 633 F2M Q V Q L V E S G G G L V Q PG G S L R L S C S A S G 634 F3 Q V Q L V E S G G G L V Q P G G S L R L SC S A S G 635 F7 Q V Q L V E S G G G V V Q P G R S L R L S C A A S G 636hv3005 Q V Q L V E S G G G V V Q P G R S L R L S C A A S G 637 F2 Q V QL V E S G G G V V Q P G R S L R L S C A A S G 638 dp-48 E V Q L V E S GG G L V Q P G G S L R L S C A A S G 639 dp-58 E V Q L V E S G G G L V QP G G S L R L S C A A S G 640 B1 Q V Q L V E S G G G V V Q P G R S L R LS C A A S G 641 B13 Q V Q L V E S G G G V V Q P G R S L R L S C A A S G642 B18 Q V Q L V E S G G G V V Q P G R S L R L S C A A S G 643 B26 Q VQ L V E S G G G V V Q P G R S L R L S C A A S G 644 B28E Q V Q L V E S GG G V V Q P G R S L R L S C A A S G 645 B29E Q V Q L V E S G G G V V Q PG R S L R L S C A A S G 646 B29M Q V Q L V E S G G G V V Q P G R S L R LS C A A S G 647 B30 Q V Q L V E S G G G V V Q P G R S L R L S C A A S G648 B32M Q V Q L V E S G G G V V Q P G R S L R L S C A A S G 649 cos-3 QV Q L V E S G G G V V Q P G R S L R L S C A A S G 650 dp-49 Q V Q L V ES G G G V V Q P G R S L R L S C A A S G 651 dp-50 Q V Q L V E S G G G VV Q P G R S L R L S C A A S G 652 P6 Q V Q L V E S G G G V V Q P G R S LR L S C A A S G 653 P9E Q V Q L V E S G G G V V Q P G R S L R L S C A AS G 654 v3-30 Q V Q L V E S G G G V V Q P G R S L R L S C A A S G 655v3-33 Q V Q L V E S G G G V V Q P G R S L R L S C A A S G 656 dp-51 E VQ L V E S G G G L V Q P G G S L R L S C A A S G 657 dp-77 E V Q L V E SG G G L V K P G G S L R L S C A A S G 658 HHG4 E V Q L V E S G G G L V KP G G S L R L S C A A S G 659 v3-21 E V Q L V E S G G G L V K P G G S LR L S C A A S G 660 v3-48 E V Q L V E S G G G L V Q P G G S L R L S C AA S G 661 DP-52 E V Q L V E S G G G L V Q P G G S L R L S C A A S G 662cos-6 E V Q L V E S G G G L V Q P G G S L R L S C A A S G 663 dp-53 E VQ L V E S G G G L V Q P G G S L R L S C A A S G 664 dp-54 E V Q L V E SG G G L V Q P G G S L R L S C A A S G 665 dp-87 E V Q L V E S G G G L VQ P G G S L R L S C A A S G 666 VH3-11 E V Q L V E S G G G L V Q P G G SL R L S C A A S G 667 JOE9  Q V Q L V Q S G G G V V Q P G R S L R L S CA A S G VH SEQ germ- CDR ID line H1 NO: VH 27 28 29 30 31 32 33 34 35 3637 38 39 40 41 42 43 44 45 46 47 48 49 594 dp-29 F T F S D H Y M D W V RQ A P G K G L E W V G 595 DP-30 F T F S D H Y M S W V R Q A Q G K G L EL V G 596 HC15-7 F T F S D H Y M S W V R Q A Q G K G L E L V G 597 VHD26F T F S D H Y M S W V R Q A Q G K G L E L V G 598 DP-31 F T F D D Y A MH W V R Q A P G K G L E W V S 599 DP-32 F T F D D Y G M S W V R Q A P GK G L E W V S 600 DP-33 F T F D D Y T M H W V R Q A P G K G L E W V S601 dp-35 F T F S D Y Y M S W I R Q A P G K G L E W V S 602 VH3-8 F T FS D Y Y M S W I R Q A P G K G L E W V S 603 yac-9 F T F S G S A M H W VR Q A S G K G L E W V G 604 dp-38 F T F S N A W M S W V R Q A P G K G LE W V G 605 LSG2 F T F S N A W M S W V R Q A P G K G L E W V G 606 LSG3F T F S N A W M S W V R Q A P G K G L E W V G 607 LSG4 F T F S N A W M SW V R Q A P G K G L E W V G 608 LSG6 F T F S N A W M N W V R Q A P G K GL E W V G 609 v3-15 F T F S N A W M S W V R Q A P G K G L E W V G 610dp-39 F T F S N H Y M S W V R Q A P G K G L E W V S 611 dp-40 F T F S NH Y T S W V R Q A P G K G L E W V S 612 dp-59 F T F S N S D M N W V H QA P G K G L E W V S 613 v3-16p F T F S N S D M N W A R K A P G K G L E WV S 614 v3-19p F T F S N S D M N W V R Q A P G K G L E W V S 615 v3-13 FT F S N Y D M N W V R Q A T G K G L E W V S 616 DP-42 F T V S S N Y M SW V R Q A P G K G L E W V S 617 dp-44 F T F S S Y A M H W V R Q A P G KG L E W V S 618 DP-45 F T F S S Y A M H W V R Q A P G K G L E W V S 619dp-47 F T F S S Y A M S W V R Q A P G K G L E W V S 620 flm F T F S S YA M H W V R Q A P G K G L E Y V S 621 P1 F T F S S Y A M H W V R Q A P GK G L E Y V S 622 v3-64 F T F S S Y A M H W V R Q A P G K G L E Y V S623 vh26 F T F S S Y A M S W V R Q A P G K G L E W V S 624 B25 F T F S SY A M H W V R Q A P G K G L E W V A 625 b32e F T F S S Y A M H W V R Q AP G K G L E W V A 626 B37 F T F S S Y A M H W V R Q A P G K G L E W V A627 B43 F T F S S Y A M H W V R Q A P G K G L E W V A 628 B48 F T F S SY A M H W V R Q A P G K G L E W V A 629 B52 F T F S S Y A M H W V R Q AP G K G L E W V A 630 B54 F T F S S Y A M H W V R Q A P G K G L E W V A631 cos-B F T F S S Y A M H W V R Q A P G K G L E W V A 632 dp-46 F T FS S Y A M H W V R Q A P G K G L E W V A 633 F2M F T F S S Y A M H W V RQ A P G K G L E Y V S 634 F3 F T F S S Y A M H W V R Q A P G K G L E Y VS 635 F7 F T F S S Y A M H W V R Q A P G K G L E W V A 636 hv3005 F T FS S Y A M H W V R Q A P G K G L E W V A 637 F2 F T F S S Y A M H W V R QA P G K G L E W V A 638 dp-48 F T F S S Y D M N W V R Q A T G K G L E WV S 639 dp-58 F T F S S Y E M N W V R Q A P G K G L E W V S 640 B1 F T FS S Y A M H W V R Q A P G K G L E W V A 641 B13 F T F S S Y A M H W V RQ A P G K G L E W V A 642 B18 F T F S S Y G M H W V R Q A P G K G L E WV A 643 B26 F T F S S Y G H H W V R Q A P G K G L E W V A 644 B28E F T FS S Y G M H W V R Q A P G K G L E W V A 645 B29E F T F S S Y G M H W V RQ A P G K G L E W V A 646 B29M F T F S S Y G M H W V R Q A P G K G L E WV A 647 B30 F T F S S Y G M H W V R Q A P G K G L E W V A 648 B32M F T FS S Y G M H W V R Q A P G K G L E W V A 649 cos-3 F T F S S Y G M H W VR Q A P G K G L E W V A 650 dp-49 F T F S S Y G M H W V R Q A P G K G LE W V A 651 dp-50 F T F S S Y G M H W V R Q A P G K G L E W V A 652 P6 FT F S S Y G M H W V R Q A P G K G L E W V A 653 P9E F T F S S Y G M H WV R Q A P G K G L E W V A 654 v3-30 F T F S S Y G M H W V R Q A P G K GL E W V A 655 v3-33 F T F S S Y G M H W V R Q A P G K G L E W V A 656dp-51 F T F S S Y S M N W V R Q A P G K G L E W V S 657 dp-77 F T F S SY S M N W V R Q A P G K G L E W V S 658 HHG4 F T F S S Y S M N W V R Q AP G K G L E W V S 659 v3-21 F T F S S Y S M N W V R Q A P G K G L E W VS 660 v3-48 F T F S S Y S M N W V R Q A P G K G L E W V S 661 DP-52 F TF S S Y V L H W V R Q A P G K G P E W V S 662 cos-6 F T F S S Y W M H WV R Q A P G K G L V W V S 663 dp-53 F T F S S Y W M H W V R Q A P G K GL V W V S 664 dp-54 F T F S S Y W M S W V R Q A P G K G L E W V A 665dp-87 F T F S S Y W M H W V R Q A P G K G L V W V S 666 VH3-11 F T F S SY W M S W V R Q A P G K G L E W V A 667 JOE9  F T F S S Y G M H W V R QA P G K G L E W V A VH SEQ germ- ID line CDR H2 NO: VH 50 51 52 52A 52B52C 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 594dp-29 R T R N K A N S Y T T E Y A A S V K G R F T I S R D 595 DP-30 L IR N K A N S Y T T E Y A A S V K G R L T I S R E 596 HC15-7 L I R N K A NS Y T T E Y A A S V K G R L T I S R E 597 VHD26 L I R N K A N S Y T T EY A A S V K G R L T I S R E 598 DP-31 G I S W . . N S G S I G Y A D S VK G R F T I S R D 599 DP-32 G I N W . . N G G S T G Y A D S V K G R F TI S R D 600 DP-33 L I S W . . D G G S T Y Y A D S V K G R F T I S R D601 dp-35 Y I . . S S S G S T I Y Y A D S V K G R F T I S R D 602 VH3-8Y I . . S S S S E Y T N Y A D S V N G R F T I S R D 603 yac-9 R I R S KA N S Y A T A Y A A S V K G R F T I S R D 604 dp-38 R I K S N T D G G TT D Y A A P V K G R F T I S R D 605 LSG2 R I E S K T D G G T T D Y A A PV K G R F T I S R D 606 LSG3 R I K S K T D G G T T D Y A A P V K G R F TI S R D 607 LSG4 R I K S K T D G G T T N Y A A P V K G R F T I S R D 608LSG6 R I K S K T D G G T T D Y A A P V K G R F T I S R D 609 v3-15 R I KS K T D G G T T D Y A A P V K G R F T I S R D 610 dp-39 Y I . . S G D SG Y T N Y A D S V K G R F T I S R D 611 dp-40 Y S . . S G N S G Y T N YA D S V K G R F T I S R D 612 dp-59 G V . . S W N G S R T H Y A D S V KG R F T I S R D 613 v3-16p G V . . S W N G S R T H Y V D S V K R R F I IS R D 614 v3-19p G V . . S W N G S R T H Y A D S V K G R F I I S R D 615v3-13 A N . . G T A G . D T Y Y P G S V K G R F T I S R E 616 DP-42 V I. Y . . S G G S T Y Y A D S V K G R F T I S R D 617 dp-44 A I . . . G TG G G T Y Y A D S V K G R F T I S R D 618 DP-45 A I . . . G T G G G T YY A D S V K G R F T I S R D 619 dp-47 A I . . S G S G G S T Y Y A D S VK G R F T I S R D 620 flm A I . . S S N G G S T Y Y A D S V K G R F T IS R D 621 P1 A I . . S S N G G S T Y Y A D S V K G R F T I S R D 622v3-64 A I . . S S N G G S T Y Y A N S V K G R F T I S R D 623 vh26 A I .. S G S G G S T Y Y G D S V K G R F T I S R D 624 B25 V I . . S Y D G SN K Y Y T D S V K G R F T I S R D 625 b32e V I . . S Y D G S N K Y Y A DS V K G R F T I S R D 626 B37 V I . . S Y D G S N K Y Y A D S V K G R FT I S R D 627 B43 V I . . S Y D G S N K Y Y A D S V K G R F T I S R D628 B48 V I . . S Y D G S N K Y Y A D S V K G R F T I S R D 629 B52 V I. . S Y D G S N K Y Y A D S V K G R F T I S R D 630 B54 V I . . S Y D GS N K Y Y A D S V K G R F T I S R D 631 cos-B V I . . S Y D G S N K Y YA D S V K G R F T I S R D 632 dp-46 V I . . S Y D G S N K Y Y A D S V KG R F T I S R D 633 F2M A I . . S S N G G S T Y Y A D S V K G R F T I SR D 634 F3 A I . . S S N G G S T Y Y A D S V K G R F T I S R D 635 F7 VI . . S Y D G S N K Y Y A D S V K G R F A I S R D 636 hv3005 V I . . S YD G S N K Y Y A D S V K G R F T I S R D 637 F2 V I . . S Y D G S N K Y YA D S V K G R F T I S R D 638 dp-48 A I . . G T A G . D T Y Y P G S V KG R F T I S R E 639 dp-58 Y I . . S S S G S T I Y Y A D S V K G R F T IS R D 640 B1 V I . . S Y D G S N K Y Y A D S V K G R F T I S R D 641 B13V I . . S Y D G S N K Y Y A D S V K G R F T I S R D 642 B18 V I . . S YD G S N K Y Y A D S V K G R F T I S R D 643 B26 V I . . S Y D G S N K YY A D S V K G R F T I S R D 644 B28E V I . . S Y D G S N K Y Y A D S V KG R F T I S R D 645 B29E V I . . S Y D G S N K Y Y A D S V K G R F T I SR D 646 B29M V I . . S Y D G S N K Y Y A D S V K G R F T I S R D 647 B30V I . . W Y D G S N K Y Y A D S V K G R F T I S R D 648 B32M V I . . S YD G S N K Y Y A D S V K G R F T I S R D 649 cos-3 F I . . R Y D G S N KY Y A D S V K G R F T I S R D 650 dp-49 V I . . S Y D G S N E Y Y A D SV K G R F T I S R D 651 dp-50 V I . . W Y D G S N K Y Y A D S V K G R FT I S R D 652 P6 V I . . W Y D G S N K Y Y A D S V K G R F T I S R D 653P9E V I . . S Y D G S N K Y Y A D S V K G R F T I S R D 654 v3-30 V I .. S Y D G S N K Y Y A D S V K G R F T I S R D 655 v3-33 V I . . W Y D GS N K Y Y A D S A K G R F T I S R D 656 dp-51 Y I . . S S S S S T I Y YA D S V K G R F T I S R D 657 dp-77 S I . . S S S S S Y I Y Y A D S V KG R F T I S R D 658 HHG4 S I . . . S S S S Y I Y Y A D S V K G R F T I SR D 659 v3-21 S I . . S S S S S Y I Y Y A D S V K G R F T I S R D 660v3-48 Y I . . S S S S S T I Y Y A D S V K G R F T I S R D 661 DP-52 A IG . . . T G G D T Y Y A D S V M G R F T I S R D 662 cos-6 R I . . N S DG S S T S Y A D S V K G R F T I S R D 663 dp-53 R I . . N S D G S S T SY A D S V K G R F T I S R D 664 dp-54 N I . . K Q D G S E K Y Y V D S VK G R F T I S R D 665 dp-87 R I . . N S D G S S T S Y A D S M K G Q F TI S R D 666 VH3-11 N I . . K Q D G S E K Y Y V D S V K G R F T I S R D667 JOE9  F I . . R I D G S N R I Y A D S V K G R F T I S R D VH SEQgerm- ID line NO: VH 73 74 75 76 77 78 79 80 81 82 82A 82B 82C 83 84 8586 87 88 89 90 91 92 93 94 594 dp-29 D S K N S L Y L Q M N S L K T E D TA V Y Y C A R 595 DP-30 D S K N T L Y L Q M S S L K T E D L A V Y Y C AR 596 HC15-7 D S K N T L Y L Q M S S L K T E D L A V Y Y C A R 597 VHD26D S K N T L Y L Q M S S L K T E D L A V Y Y C A R 598 DP-31 N A E N S LY L Q M N S L R A E D T A L Y Y C A K 599 DP-32 N A K N S L Y L Q M N SL R A E D T A L Y H C A R 600 DP-33 N S K N S L Y L Q M N S L R T E D TA L Y Y C A K 601 dp-35 N A K N S L Y L Q H N S L R A E D T A V Y Y C AR 602 VH3-8 N A K N S L Y L Q N N S L R A E D T A V Y Y C A R 603 yac-9D S K N T A Y L Q M N S L K T E D T A V Y Y C T R 604 dp-38 D S K N T LY L Q M N S L K T E D T A V Y Y C T T 605 LSG2 D S K N T L Y L Q M N S LK T E D T A V Y Y C T T 606 LSG3 D S K N T L Y L Q M N S L K T E D T A VY Y C T T 607 LSG4 D S K N T L Y L Q M N S L K T E D T A V Y Y C T T 608LSG6 D S K N T L Y L Q M N S L K T E D T A V Y Y C T T 609 v3-15 D S K NT L Y L Q M N S L K T E D T A V Y Y C T T 610 dp-39 N A N N S P Y L Q MN S L R A E D T A V Y Y C V K 611 dp-40 N A K N S L Y L Q M N S L R A ED T A V Y Y C V K 612 dp-59 N S R N T L Y L Q T N S L R A E D T A V Y YC V R 613 v3-16p N S R N S L Y L Q K N R R R A E D M A V Y Y C V R 614v3-19p N S R N F L Y Q Q M N S L R P E D M A V Y Y C V R 615 v3-13 N A KN S L Y L Q M N S L R A E G D T A V Y C A R 616 DP-42 N S K N T L Y L QM N S L R A E D T A V Y Y C A R 617 dp-44 N A K N S L Y L Q M N S L R AE D M A V Y Y C A R 618 DP-45 N A K N S L Y L Q M N S L R A E D M A V YY C A R 619 dp-47 N S K N T L Y L Q M N S L R A E D T A V Y Y C A K 620flm N S K N T L Y V Q M S S L R A E D T A V Y Y C V K 621 P1 N S K N T LY V Q M S S L R A E D T A V Y Y C V K 622 v3-64 N S K N T L Y L Q M G SL R A E D M A V Y Y C A R 623 vh26 N S K N T L Y L Q M N S L R A E D T AV Y Y C A K 624 B25 N S K N T L Y L Q M N S L R A E D T A V Y Y C A R625 b32e N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 626 B37 N S KN T L Y L Q M S S L R A E D T A V Y Y C A R 627 B43 N S K N T L Y L Q MN S L R A E D T A V Y Y C A R 628 B48 N S K N T L Y L Q M N S L R A E DT A V Y Y C A R 629 B52 N S K N T L Y L Q M N S L R A E D T A V Y Y C AR 630 B54 N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 631 cos-B NS K N T L Y L Q M N S L R A E D T A V Y Y C A R 632 dp-46 N S K N T L YL Q M N S L R A E D T A V Y Y C A R 633 F2M N S K N T L Y V Q M S S L RA E D T A V Y Y C V K 634 F3 N S K N T L Y V Q M N S L R A E D T A V Y YC V R 635 F7 N S K H T L Y L Q M N S L R A E D T A V Y Y C A R 636hv3005 N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 637 P2 N S E NT L Y L Q M N S L R A E D T A V Y Y C A K 638 dp-48 N A K N S L Y L Q MN S L R A G D T A V Y Y C A R 639 dp-58 N A K N S L Y L Q M N S L R A ED T A V Y Y C A R 640 B1 N S K N T L Y L Q M N S L R L R A R L C I T V RE 641 B13 N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 642 B18 N SK N T L Y L Q M N S L R A E D T A V Y Y C A R 643 B26 N S K N T L Y L QM N S L R A E D T A V Y Y C A R 644 B28E N S K N T L Y L Q M N S L R A ED T A V Y Y C A R 645 B29E N S K N T L Y L Q M N S L R A E D T A V Y Y CA R 646 B29M N S K H T L Y L Q M N S L R A E D T A V Y Y C A R 647 B30 NS K N T L Y L Q M N S L R A E D T A V Y Y C A R 648 B32M N S K N T L Y LQ M N S L R A E G T A V Y Y C A R 649 cos-3 N S K N T L Y L Q M N S L RA E D T A V Y Y C A K 650 dp-49 N S K N T L Y L Q M N S L R A E D T A VY Y C A K 651 dp-50 N S K N T L Y L Q M N S L R A E D T A V Y Y C A R652 P6 N S K N T L Y L Q M N S L R A E D T A V Y Y C A K 653 P9E N S K NT L Y L Q M N S L R A E D T A V R K - - - 654 v3-30 N S K N T L Y L Q MN S L R A E D T A V Y Y C A R 655 v3-33 N S T N T L F L Q M N S L R A ED T A V Y Y C A R 656 dp-51 N A K N S L Y L Q M N S L R D E D T A V Y YC A R 657 dp-77 N A K N S L Y L Q M N S L R A E D T A V Y Y C A R 658HHG4 N A K N S L Y L Q M N S L R A E D T A V Y Y C A R 659 v3-21 N A K NS L Y L Q M N S L R A E D T A V Y Y C A R 660 v3-48 N A K N S L Y L Q MN S L R A E D T A V Y Y C A R 661 DP-52 N A K K S L Y L Q M N S L I A ED M A V Y Y C A R 662 cos-6 N A K N T L Y L Q M N S L R A E D T A V Y YC A R 663 dp-53 N A K N T L Y L Q M N S L R A E D T A V Y Y C A R 664dp-54 N A K N S L Y L Q M N S L R A E D T A V Y Y C A R 665 dp-87 N A KN T L Y L Q M N S L R A E D M A V Y Y C T R 666 VH3-11 N A K N S L Y L QM N S L R A E D T A V Y Y C A R 667 JOE9 N S K N T L Y L Q M K S L R A ED T A V Y Y C T T VH Vλ1 Family Germline Amino Acid SequencesNumbering according to Kabat. (Joe9 VL included for comparison) SEQ CDRID L1 NO: gene* VL 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2122 23 24 25 668 1b DPL5 Q S V L T Q P P S V S A A P G Q K V T I S C S GS 669 1d DPL4 Q S V L T Q P P S V S A A P G Q K V T I S C S G S 670 1cDPL2 Q S V L T Q P P S A S G T P G Q R V T I S C S G S 671 1g DPL3 Q S VL T Q P P S A S G T P G Q R V T I S C S G S 672 1a DPL1 Q S V L T Q P PS V S E A P R Q R V T I S C S G S 673 1f DPL9 Q S V L T Q P P S V S G AP G Q R V T I S C T G S 674 1e DPL8 Q S V V T Q P P S V S G A P G Q R VT I S C T G S 675 JOE9  H T V L T Q P P S V S G T P G Q R V T I S C S GG VL SEQ ID CDR L1 NO: gene* VL 26 27A 27B 27C 28 29 30 31 32 33 34 3536 37 38 39 40 41 42 43 44 45 46 47 48 668 1b DPL5 S S N I G N N Y . V SW Y Q Q L P G T A P K L L I 669 1d DPL4 S S D M G N Y A . V S W Y Q Q LP G T A P K L L I 670 1c DPL2 S S N I G S N T . V N W Y Q Q L P G T A PK L L I 671 1g DPL3 S S N I G S N Y . V Y W Y Q Q L P G T A P K L L I672 1a DPL1 S S N I G N N . A V N W Y Q Q L P G K A P K L L I 673 1fDPL9 S S N I G A G Y V V H W Y Q Q L P G T A P K L L I 674 1e DPL8 S S NI G A G Y D V H W Y Q Q L P G T A P K L L I 675 JOE9  R S N I G S N T .V K W Y Q Q L P G T A P K L L I VL SEQ ID CDR L2 NO: gene* VL 49 50 5152 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 668 1bDPL5 Y D N N K R P S G I P D R F S G S K S G T S A T L 669 1d DPL4 Y E NN K R P S G I P D R F S G S K S G T S A T L 670 1c DPL2 Y S N N Q R P SG V P D R F S G S K S G T S A S L 671 1g DPL3 Y R N N Q R P S G V P D RF S G S K S G T S A S L 672 1a DPL1 Y Y D D L L P S G V S D R F S G S KS G T S A S L 673 1f DPL9 Y G N S N R P S G V P D Q F S G S K S G T S AS L 674 1e DPL8 Y G N S N R P S G V P D R F S G S K S G T S A S L 675JOE9  Y G N D Q R P S G V P D R F S G S K S G T S A S L VL SEQ ID CDR L3NO: gene* VL 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 9394 95 96A 96B 668 1b DPL5 G I T G L Q T G D E A D Y Y C G T W D S S L SA 669 1d DPL4 G I T G L W P E D E A D Y Y C L A W D T S P R A 670 1cDPL2 A I S G L Q S E D E A D Y Y C A A W D D S L N G 671 1g DPL3 A I S GL R S E D E A D Y Y C A A W D D S L S G 672 1a DPL1 A I S G L Q S E D EA D Y Y C A A W D D S L N G 673 1f DPL9 A I T G L Q S E D E A D Y Y C KA W D N S L N A 674 1e DPL8 A I T G L Q A E D E A D Y Y C Q S Y D S S LS G 675 JOE9  A I T G V Q A E D E A D Y Y C Q S Y D S S L R G VL*Williams, J M B, 1996, 264, 220-232

TABLE 2 H3 L3 RB PHA IFN SEQ SEQ assay assay gamma ID ID IC50 IC50 IC50Clone NO: H3 NO: L3 koff (M) (M) (M) Joe9   77 SGSYDY 110 QSYDSSLRGSRV1.00E−01 1.50E−06 1.00E− wt 06 Joe9    77 SGSYDY 110 QSYDSSLRGSRV 5.00E−wt 07 IgG1 70-1  78 HGSHDN 110 Joe9 wt 1.34 e−2 2.00E− 07 70-1   78HGSHDN 110 Joe9 wt 2.00E− IgG1 07 70-2  79 HGSYDY 110 Joe9 wt 3.30E−02 3-5.0E− 7 70-7  80 RRRSNY 110 Joe9 wt 1.29E−01 3-5.0E− 7 70-13  81SGSIDY 110 Joe9 wt 7.20E−02 3-5.0E− 7 78-34  77 wt 111 QSYDRGFTGSRV1.64 e−2 2.00E−07 6.00E− 07 78-25  77 wt 112 QSYDSSLRGSRV 5.00E−02 78-28 77 wt 112 QSYDSSLRGSRV 4.66E−02 78-35  77 wt 113 QSYDSSLTGSRV 4.99E−024.00E−07 79-1  77 wt 114 QSYDSSLWGSRV 2.00E−07 6.00E− 07 101-14  79 70-2111 78-34 7.52E−03 101-9  79 70-2 113 78-35 8.54E−03 101-19  81 70-13111 78-34 4.56E−02 101-8  81 70-13 111 78-34 1.01E−02 101-4  81 70-13113 78-35 9.76E−03 101-5  81 70-13 113 78-35 4.45E−02 101-11   78 70-1111 78-34 4.5 e−3 3.00E− (12) 08 101-11   78 70-1 111 78-34 1.60E−09IgG1 26-1   78 70-1 114 79-1 7.4 e−3 6.00E− (2, 3) 08 136-9  82 HGSHDD115 QTYDISESGSRV 3.20E−03 136-10  82 HGSHDD 116 QSYDRGFTGSRV 1.40E−032.00E−09 136-14  83 HGSHDN 117 QTYDRGFTGSRV 1.10E−03 3.00E−10 1.00E− 07136-15  83 HGSHDN 118 QTYDKGFTGSSV 7.4 e−4 1.00E−10 2.00E− 09 136-15  83 HGSHDN 118 QTYDKGFTGSSV 4.60E−04 6.00E− germ- 09 line 136-16  83HGSHDN 119 QSYDRRFTGSRV 6.10E−04 3.00E−10 5.00E− 09 136-17  83 HGSHDN120 QSYDWNFTGSRV 2.90E−05 2.00E−09 7.00E− 09 136-18  83 HGSHDN 121QSYDRGFTGSRV 1.10E−03 8.00E−10 136-21  83 HGSHDN 122 QSYDNGFTGSRV4.20E−04 2.00E−09 136-24  83 HGSHDN 123 QSYDNAVTASKV 8.90E−04 1.00E−09101-11  84 TT_HGSHDN_WGQG 124 QSYDRGFTGSRV 4.5 × 10−3   2 × 10−9 2.00E−08 136-  85 AK ...... .... 124 QSYDRGFTGSRV 4.00E−10 15M1 149-4  86.. ...... .S.. 124 ............ 1.37 × 10−3   8 × 10−11 3.00E− 09 149-5 87 .. .....T .... 125 QSYDSSLWGTRV 1.02 × 10−3 1.2 × 10−10 3.00E− 09149-6  84 .. ...... .... 124 ............ 2.73 × 10−3   6 × 10−10 2.00E−09 149-7  84 .. ...... .... 126 .....D...... 1.13 × 10−3   9 × 10−103.00E− 09 149-8  88 K. ...... .... 2.33 × 10−3   3 × 10−9 149-9  89K. ...... ..H. 127 ...E......M. 3.54 × 10−3 1.8 × 10−10 149-11  90.. ...... .S.. 128 ....N....A.. 1.43 × 10−2   2 × 10−10 4.00E− 09 149-12 84 .. ...... .... 3.73 × 10−3 neutralising 149-13  84 .. ...... ....2.22 × 10−3   5 × 10−10 149-14  91 .. .R..N. .... 1.5 × 10−10 6.00E− 09 92 TT_HGSHDN 124 QSYDRGFTGSRV 156-1  93 .. .....T 126 .....D......5.00E−03 156-2  93 .. .....T 129 .....R...... 156-3  93 .. .....T 128....N....A.. 9.00E−03 156-4  93 .. .....T 127 ...E.....SM. 156-5  93.. .....T 130 .T..K.....S. 156-6  92 .. ...... 126 .....D...... 3.00E−03156-7  92 .. ...... 129 .....R...... 156-8  92 .. ...... 128....N....A.. 156-9  92 .. ...... 127 ...E.....SM. 156-10  92 .. ......130 .T..K.....S. 156-11  94 .K ...... 126 .....D...... 156-12  94.K ...... 129 .....R...... 156-13  94 .K ...... 128 ....N....A.. 156-14 94 .K ...... 127 ...E.....SM. 156-15  94 .K ...... 130 .T..K.....S.156-16  93 .. .....T 124 ............ 156-17  92 .. ...... 125....SSLW.T.. 6.00E−03 156-18  93 .. .....T 125 ....SSLW.T..  92TT_HGSHDN 124 QSYDRGFTGSRV 103-1  95 .. Q.R... 124 ............ 2.9 ×10−3 103-2  96 K. R.R... 130 .T..K.....S. 7.3 × 10−4 7.00E−11 1.00E− 09103-3  97 .. .....K 124 ............ 2.5 × 10-3 103-6 131 .....D...T..4.5 × 10−4 103-7  98 .. .....D 131 .....D...T.. 3.7 × 10−4 1.40E−101.00E− 09 103-8  99 K. ...... 130 .T..K.....S. 3.3 × 10−4 6.00E−111.50E− 09 103- 100 KT_HGSHDN 132 QSYDRGFTGSMV 6.7 e−4 4.00E−11 1.20E−14 & 9 09 103- 100 KT_HGSHDN 133 QTYDKGFTGSSV 5.3 e−4 1.50E− 8 & 2 09103-4 101 TT_HGSHDN 134 QSYDRGFTGARV 1.6 e−4 8.60E−11 9.00E− 10 103-152101 TT_HGSHDN 135 QSYERGFTGARV 8.60E−11 102 TT_SGSYDY 136 QSYDRGFTGSRVF170-1 102 .. ...... 137 .........FK.. 2.35E−03 170-2 102 .. ...... 138.......VSAY.. 8.80E−04 170-3 102 .. ...... 139 ......L.VTK.. 1.11E−03170-4 102 .. ...... 140 ......Y.A.... 8.11E−04 170-7 102 .. ...... 141..........K.. 5.30E−04 170-11 102 .. ...... 142 ......L..F... 4.40E−04170-13 102 .. ...... 143 .........YK.. 1.59E−03 170-15 102 .. ...... 144......L..Y.L. 4.43E−03 170-19 103 .. H..H.N 145 ........DYK.. 1.00E−03170-21 104 .. H..Q.N 146 .........P.L. 3.69E−03 170-22 102 .. ...... 147......L...... 5.60E−04 170-23 103 .. H..H.N 148 .........A..W 1.00E−032.00E−10 170-24 104 .. H..Q.N 149 .........Y... 2.80E−04 5.00E−10 170-35105 A. H..Q.N 136 ............. 1.00E−05 170-38 150 .........P...2.10E−04 170-39 151 ......M.S.... 2.79E−03 170-36  83 HGSHDN 152QSYDRDSTGSRVF 4.00E−04 2.00E−10 170-25 106 HGSQDT 153 QSYDSSLRGSRVF5.00E−04 5.00E−11 106 SGSYDY 136 QSYDRGFTGSRVF 73-B1 107 SGSYDY 154H...SD....... 3.25E−03 >1E−8 73-B2 107 SGSYDY 155 H.SES........ 2.07E−0373-B6 107 SGSYDY 156 H...NR....... 2.51E−03 >1E−8 73-C1 107 SGSYDY 157H...SR....... 2.71E−03 >1E−8 73-C2 107 SGSYDY 158 ....SE....... 3.79E−0373-C6 107 SGSYDY 159 ....T........ 3.96E−03 73-D1 107 SGSYDY 160H...S........ 3.99E−03 73-D2 107 SGSYDY 161 ....T........ 3.56E−03 73-D4107 SGSYDY 162 H...TK....... 5.36E−03 73-D5 107 SGSYDY 163 H.S.S........3.57E−03 73-E3 107 SGSYDY 164 ....SD....... 4.98E−03 73-E6 107 SGSYDY165 H..ES........ 4.17E−03 73-F3 107 SGSYDY 166 ....APWS..... 7.08E−0373-F5 107 SGSYDY 167 ...DSD....K.. 3.74E−03 73-G2 107 SGSYDY 168HTN.S........ 3.98E−03 73-G3 107 SGSYDY 169 H...TR....... 3.50E−03 73-G4107 SGSYDY 170 ....MR....... 6.58E−03 73-G5 107 SGSYDY 171 H.S.SDS......6.01E−03 73-G6 107 SGSYDY 172 ...NTD....... 6.30E−03 73-H2 107 SGSYDY173 ....S........ 5.93E−03 73-F6 107 SGSYDY 174 H...M........ 5.87E−0373-H3 107 SGSYDY 175 H...N........ 6.85E−03 73-C5 107 SGSYDY 176H.H..D....... 4.84E−03 73-B7 108 HGSQDN 177 QSYDSSLRGSRV 2.50E−037.00E−09 136 QSYDRGFTGSRVF M2 A2  83 HGSHDN 178 ......IH..... 4.00E−02M2 A4  83 HGSHDN 179 ....S..P..... 8.49E−03 M2 A5  83 HGSHDN 180....I.S...... 4.01E−02 M2 B1  83 HGSHDN 181 ....S.L...... 7.97E−03 M2 B3 83 HGSHDN 182 ....I.M...... 4.60E−02 M2 B4  83 HGSHDN 183 ....I.L......4.42E−02 M2 B5  83 HGSHDN 184 ....S.V...... 8.38E−03 M2 B6  83 HGSHDN185 ......L.A.... 2.81E−02 M2 C2  83 HGSHDN 181 ....S.L...... 4.85E−02M2 C3  83 HGSHDN 186 ....T.L...... 4.62E−02 M2 C4  83 HGSHDN 181....S.L...... 8.16E−03 M2 C5  83 HGSHDN 187 ....TAL...... 4.71E−02 M2 D1 83 HGSHDN 188 ....IR....... 3.71E−02 M2 D2  83 HGSHDN 189 ....IRS......3.85E−02 M2 D3  83 HGSHDN 190 ....NRL...... 3.33E−02 M2 D4  83 HGSHDN191 ...ETS....... 5.81E−02 M2 D5  83 HGSHDN 192 ....SSS...... 5.18E−02M2 D6  83 HGSHDN 193 ....S...A.... 5.01E−02 M2 E1  83 HGSHDN 194.T..K.....S.. 5.32E−02 M2 E2  83 HGSHDN 195 ....N........ 4.77E−02 M2 E6 83 HGSHDN 196 ....T...K.... 9.77E−03 M2 F1  83 HGSHDN 197 ....SDV......6.16E−02 M2 H5  83 HGSHDN 198 ....A........ 9.90E−03 124 QSYDRGFTGSRV A5 83 HGSHDN 199 ......THPSML 1.12E−03 A12  83 HGSHDN 200 ......TTPRPM1.43E−03 A4  83 HGSHDN 201 ......RNPALT 1.47E−03 A6  83 HGSHDN 202......THPWLH 1.87E−03 A10  83 HGSHDN 203 ......NSPATV 1.87E−03 A11  83HGSHDN 204 ......TFPSPQ 2.07E−03 C2  83 HGSHDN 205 ......LNPSAT 2.23E−03A8  83 HGSHDN 206 ......KSNKML 2.37E−03 B8  83 HGSHDN 207 ......HTAHLY2.40E−03 C6  83 HGSHDN 208 ......QTPSIT 2.42E−03 A3  83 HGSHDN 209......YPRNIL 2.51E−03 B11  83 HGSHDN 210 ......ITPGLA 2.95E−03 B5  83HGSHDN 211 ......QPHAVL 3.04E−03 C10  83 HGSHDN 212 ......NSPIPT3.10E−03 C4  83 HGSHDN 213 ......TPNNSF 3.23E−03 C3  83 HGSHDN 214....S.VDPGPY 3.34E−03 B2  83 HGSHDN 215 ......RPRHAL 3.61E−03 A2  83HGSHDN 216 ......PYHPIR 3.80E−03 C5  83 HGSHDN 217 ......PHTQPT 3.91E−03A7  83 HGSHDN 218 ......HNNFSP 3.95E−03 C9  83 HGSHDN 219 ......PTHLPH3.97E−03 B3  83 HGSHDN 220 ......TPSYPT 4.12E−03 C8  83 HGSHDN 221....S.TSNLLP 5.36E−03 B7  83 HGSHDN 222 ......DSNHDL 5.45E−03 A1  83HGSHDN 223 ......LPRLTH 5.66E−03 C7  83 HGSHDN 224 ......IPTSYL 5.83E−03C12  83 HGSHDN 225 ......LRVQAP 5.85E−03 B10  83 HGSHDN 226 ......LSDSPL6.04E−03 B6  83 HGSHDN 227 ....S.SLRRIL 7.58E−03 A9  83 HGSHDN 228......PARTSP 7.98E−03 B9  83 HGSHDN 229 ......RAAHPQ 8.66E−03 124QSYDRGFTGSRV 177-D7  83 HGSHDN 230 ......TQPABI 4.07E−04 177-G6  83HGSHDN 231 ......THPTMI 5.50E−04 177-D9  83 HGSHDN 232 ......RIPABT6.32E−04 177-C6  83 HGSHDN 233 ......THPVPA 7.94E−04 177-H5  83 HGSHDN234 ......SBPIPA 1.32E−03 177-H9  83 HGSHDN 235 ......THPVPA 1.58E−03177-H10  83 HGSHDN 236 ......THPTMY 3.44E−03 144-F1  83 HGSHDN 237......HHYTTF 5.80E−04 43-E3  83 HGSHDN 238 ......SHPAAE 8.00E−04 43-E9 83 HGSHDN 239 ......TIPSIE 8.00E−04 43-G2  83 HGSHDN 240 ......SSPAIM7.00E−04 43-G3  83 HGSHDN 241 ......IWPNLN 9.00E−04 31-A6  83 HGSHDN 242......THPNLN 5.00E−04 31-B5  83 HGSHDN 243 ......THPSIS 5.00E−04 124QSYDRGFTGSRV Y17  83 HGSHDN 244 QSYDRGSAPMIN 8.90E−05 4.50E−10 >1E−8 Y19 83 HGSHDN 245 QSYDRGHHPAMS 2.26E−04 3.00E−11 >1E−8 Y38  83 HGSHDN 246......THPSIT 5.08E−04 5.50E−11 2.60E− 09 Y45  83 HGSHDN 247 ......TDPAIV6.17E−04 4.00E−11 4.30E− 09 Y61  83 HGSHDN 248 ......THPALL 2.75 e−44E−11 1.40E− 10 Y61   83 HGSHDN 248 ......THPALL 1.50E−04 1.60E−111.30E− IgG 10 Y61    83 HGSHDN 248 ......THPALL 1.50E−04 1.60E−11 1.30E−1.60E− IgG 10 10 germ- line Y139  83 HGSHDN 249 ......SHPALT 5.92E−043E−11 4.50E− 10 Y139   83 HGSHDN 249 ......SHPALT 1.00E− IgG1 09 Y174 83 HGSHDN 250 ......TTPAPE 7.55E−04 6E−11 2.00E− 09 Y177  83 HGSHDN 251......SHPTLI 6.61E−04 5E−11 1.00E− 09 A5  83 HGSHDN 252 ......THPSML4.50E−04 6.60E−11 A12  83 HGSHDN 253 ......TTPRPM 5.57E−04 2.50E−10 D9 83 HGSHDN 254 ......RLPAQT 8.21E−04 3.5E−09 >> G6  83 HGSHDN 255......THPLTI 5.08E−04 1E−10 1.00E− 09 G6   83 HGSHDN 255 ......THPLTI1.00E− IgG1 09 C6  83 HGSHDN 256 QSYDRGQTPSIT 1.07E−03 3.5E−10 1.00E− 08Y55  83 HGSHDN 257 QSYDRGTHFQMY 1.06E−03 1.40E−10 >1E−8 A4  83 HGSHDN258 QSYDRGRNPALT 6.30E−04 2.50E−10 AO3  83 HGSHDN 259 QSYDRGTHPLTM3.04E−04 3.00E−11 4.00E− 10 AO3   83 HGSHDN 260 QSYDRGTHPLTM 3.04 e−42.90E−11 3.80E− IgG1 10 AO3    83 HGSHDN 260 QSYDRGTHPLTM 2.50E−043.50E−11 1.75E− IgG 10 germ- line 99-B11  83 HGSHDN 261 QSYDSGYTGSRV5.40E−03 99-C11  83 HGSHDN 262 QSYDSGFTGSRV 5.70E−03 99-H4  83 HGSHDN263 QSYDSRFTGSRV 4.80E−03 99-E9  83 HGSHDN 262 QSYDSGFTGSRV 5.40E−0399-H7  83 HGSHDN 264 QSYPDGTPASRV 3.30E−03 99-H11  83 HGSHDN 265QSYSTHMPISRV 4.90E−03 99-F6  83 HGSHDN 266 QSYDSGSTGSRV 4.90E−03 99-F7 83 HGSHDN 267 QSYPNSYPISRV 4.80E−03 99-F8  83 HGSHDN 268 QSYIRAPQQV3.70E−03 99-F11  83 HGSHDN 262 QSYDSGFTGSRV 5.40E−03 99-G7  83 HGSHDN269 QSYLKSRAFSRV 4.80E−03 99-G11  83 HGSHDN 270 QSYDSRFTGSRV 4.30E−03124 QSYDRGFTGSRV L3.3R3M-  83 HGSHDN 271 ......FTGSMV 5.46E+00 B1L3.3R3M-  83 HGSHDN 272 ......FTGSMV 5.51E+00 B3 L3.3R3M-  83 HGSHDN 273......FTGFDG 6.17E+00 C6 L3.3R3M-  83 HGSHDN 274 ......TAPALS 4.99E+00F9 L3.3R3M-  83 HGSHDN 275 ......SYPALR 5.55E+00 G8 L3.3R3M-  83 HGSHDN276 ......NWPNSN 5.69E+00 H6 L3.3R3M-  83 HGSHDN 277 ......TAPSLL5.35E+00 H10 L3.3R3M-  83 HGSHDN 278 ......FTGSMV 5.37E+00 A3 L3.3R3M- 83 HGSHDN 279 ......TTPRIR 4.99E+00 F8 L3.3R3M-  83 HGSHDN 280......FTGSMV 4.21E+00 G1 L3.3R3M-  83 HGSHDN 281 ......FTGSMV 4.24E+00G7 L3.3R3M-  83 HGSHDN 282 ......MIPALT 3.95E+00 H11 Y61-L94N 109CKT HGSHDN 283 QSYDRNTHPALL 8.00E− 11 Y61-L94F 109 CKT HGSHDN 284QSYDRFTHPALL 6.00E− 11 Y61-L94Y 109 CKT HGSHDN 285 QSYDRYTHPALL 2.00E−112.00E− 11 Y61-L94Y  109 CKT HGSHDN 285 QSYDRYTHPALL 1.27E−04 6.00E−115.00E− 4.00E− IgG 11 11 Y61-L50Y 109 CKT HGSHDN 286 QSYDRGTHPALL2.00E−11 2.00E− 11 Y61-  109 CKT HGSHDN 286 QSYDRGTHPALL 6.98E−05 2.00E−3.00E− L50Y* 11 11 IgG Y61- 109 CKT HGSHDN 286 QSYDRGTHPALL 2.99E−056.00E− 2.00E− L50Y- 11 11 H31E** IgG Y61- 109 CKT HGSHDN 287QSYDRYTHPALL 4.64E−05 1.00E− 1.00E− L50Y- 11 11 H31E- L94Y**  IgG J695 109 CKT HGSHDN 287 QSYDRYTHPALL 5.14E−05 5.00E−11 1.00E− 5.00E− (Y61- 1112 L94Y- L50Y  IgG*) *CDR L2: L50G TO Y **CDR L2: L50G TO Y; CDR H1:H31S TO E

TABLE 3 CDR H1 CDR H2 Kabat Number 27 28 29 30 31 32 33 34 35 50 51 5252A 53 54 55 Y61 VH F T F S S Y G M H F I R Y D G S Contact x x x x x xx x x x Positions Hypermutation x x x x Positions CDR H2 CDR H3Kabat Number 56 57 58 59 60 61 62 63 64 65 95 96 97 98 101 102 Y61 VH NK Y Y A D S V K G H G S H D N Contact x x x x x x x PositionsHypermutation x x Positions CDR L1 CDR L2 Kabat Number 24 25 26 27 27A27B 28 29 30 31 32 33 34 50 51 52 Y61VL S G G R S N I G S N T V K G N DContact x x x x x x Positions Hypermutation x x x Positions CDR L2CDR L3 Kabat Number 53 54 55 56 89 90 91 92 93 94 95 95A 95B 95C 96 97Y61VL Q R P S Q S Y D R G T H P A L L Contact x x x x x x x PositionsHypermutation x x Positions x contact and/or hypermutation position xcontact and/or hypermutation position mutated in Y61

TABLE 4 Neutralization Activity in the Presence of Excess Free IL-12 p40PHA assay PHA assay PHA assay SEQ IC50 (M) IC50 (M) IC50 (M) ID NO:Clone p70:p40 1:0 p70:p40 1:20 p70:p40 1:50 VH: 47 136-15 2.00E−095.00E−09 4.00E−09 VL: 48 VH: 51 149-5 6.50E−09 7.00E−09 4.00E−09 VL: 52VH: 53 149-6 9.00E−10 1.00E−09 1.00E−09 VL: 54 VH: 84 149-7 3.50E−092.50E−09 4.00E−09 VL: 126 VH: 23 Y61 IgG 1.80E−10 1.80E−10 VL: 24 VH: 65AO3 IgG1 2.50E−10 2.20E−10 VL: 66 VH: 31 J695 1.00E−11 3.50E−11 VL: 32

EXAMPLES Example 1 Isolation of Anti-IL-12 Antibodies A. Screening forIL-12 Binding Antibodies

Antibodies to hIL-12 were isolated by screening three separate scFvphage display libraries prepared using human VL and VH cDNAs from mRNAderived from human tonsils (referred to as scFv 1), tonsil andperipheral blood lymphocytes (PBL) (referred to as scFv 2), and bonemarrow-derived lymphocytes (referred to as BMDL). Construction of thelibrary and methods for selection are described in Vaughan et al. (1996)Nature Biotech. 14: 309-314.

The libraries were screened using the antigens, human IL-12 p70 subunit,human IL-12 p40 subunit, chimaeric IL-12 (mouse p40/human p35), mouseIL-12, biotinylated human IL-12 and biotinylated chimaeric IL-12. IL-12specific antibodies were selected by coating the antigen ontoimmunotubes using standard procedures (Marks et al., (1991) J. Mol.Biol. 222: 581-597). The scFv library 2 was screened using either IL-12,or biotinylated-IL-12, and generated a significant number of IL-12specific binders. Five different clonotypes were selected, determined byBstN1 enzymatic digestion patterns, and confirmed by DNA sequencing. Themain clonotypes were VHDP58/VLDPL11, VHDP77/VLDPK31, VHDP47/VL andVHDP77/VLDPK31, all of which recognized the p40 subunit of IL-12.

Screening of the BMDL library with IL-12 p70 generated 3 differentclonotypes. Two of these were found to be cross-reactive clones. Thedominant clone was sequenced and consisted of VHDP35/VLDP. This clonerecognizes the p40 subunit of IL-12. Screening of the scFv library 1,using IL-12 p70, did not produce specific IL-12 antibodies.

In order to identify IL-12 antibodies which preferentially bind to thep70 heterodimer or the p35 subunit of IL-12, rather than the p40subunit, the combined scFv 1+2 library, and the BMDL library were used.To select IL-12 antibodies that recognized the p70 heterodimer or p35subunit, phage libraries were preincubated and selected in the presenceof free p40. Sequencing of isolated clones revealed 9 different antibodylineages. Subunit preferences were further analyzed by ‘micro-Friguet’titration. The supernatant containing scFv was titrated onbiotin-captured IL-12 in an ELISA and the ED₅₀ determined. Theconcentration of scFv producing 50% ED was preincubated with increasingconcentrations of free p70 or p40 (inhibitors). A decrease in the ELISAsignal on biotin-IL-12 coated plates was measured and plotted againstthe concentration of free p70 or p40. This provided the IC₅₀ for eachclone with respect to p70 and p40. If the titrations for both subunitsoverlaps, then the scFv binds to both p40 and p70. Any variation fromthis gives the degree of preference of p70 over p40.

B. Affinity Maturation of Antibody Lineage Specific for IL-12 (Joe 9)

The clones were tested for their ability to inhibit IL-12 binding to itsreceptor in an IL-12 receptor binding assay (referred to as RBA), andfor their ability to inhibit IL-12 induced proliferation of PHAstimulated human blast cells (PHA assay), described in Example 3. CloneJoe 9 had the lowest IC₅₀ value in both the RBA and the PHA assay, withan IC₅₀ value of 1×10⁻⁶ M in both assays. In addition the heavy chainvariable region (VH) of Joe 9 had the least number of changes comparedto the closest germline sequence COS-3, identified from the VBASEdatabase. Table 1 (see Appendix A) shows the V_(H)3 family of germlinesequences, of which COS-3 is a member, as well as members of V_(λ)1family of germline sequences. Therefore, Joe 9 was selected for affinitymaturation. The amino acids sequences of VH and VL of the Joe9 wild type(Joe9 wt) antibody are shown in FIG. 1A-1D.

In order to increase the affinity of Joe 9, various mutations of thecomplementarity determining region 3 (CDR3) of both the heavy and lightchains were made. The CDR3 variants were created by site-directed PCRmutagenesis using degenerate oligonucleotides specific for either theheavy chain CDR3 (referred to as “H3”) or the light chain CDR3 (referredto as “L3”), with an average of three base substitutions in each CDR3(referred to as “spike”). PCR mutagenesis of the heavy chain CDR3 wasperformed using the degenerate heavy chain oligonucleotide containing arandom mixture of all four nucleotides,5′TGTCCCTTGGCCCCA(G)(T)(A)(G)(T)(C)(A)(T)(A)(G)(C)(T)(C)(C)(C)(A)(C)(T)GGTCGTACAGTAATA 3′ (SEQ ID NO: 580), and oligonucleotide pUC Reverse TagGAC ACC TCG ATC AGC GGA TAA CAA TTTCAC ACA GG (SEQ ID NO: 581) togenerate a repertoire of heavy chain CDR3 mutants. The parent lightchain was amplified using Joe 9 reverse oligonucleotide (5′TGG GGC CAAGGG ACA3′ (SEQ ID NO:582) and the fdteteseq 24+21 oligonucleotide(5′-ATT CGT CCT ATA CCG TTC TAC TTT GTC GTC TTT CCA GAC GTT AGT-3′ (SEQID NO: 583).

Complementarity between the two PCR products was used to drive annealingof the two fragments in a PCR assembly reaction and the full lengthrecombined scFv library was amplified with pUC Reverse Tag (SEQ ID NO:581) and fdTag 5′-ATT CGT CCT ATA CCG TTC-3′ (SEQ ID NO: 584). PCRmutagenesis of the light chain was performed using the light chainoligonucleotide containing a mixture of all four nucleotides5′GGTCCCAGTTCCGAAGACCCTCGAACC(C)(C)(T)(C)(A)(G)(G)(C)(T)(G)(C)(T)(G)(T)(C)ATATGACTGGCAGTAATAGTCAGC 3′ (SEQ ID NO: 585), and Joe9 reverse oligonucleotide 5′TGG GGC CAA GGG ACA3′ (SEQ ID NO: 586) toproduce a repertoire of light chain CDR3 mutants. The parent heavy chainwas amplified with pUC Reverse Tag (SEQ ID NO: 581) and HuJH3FORoligonucleotide 5′TGAAGAGACGGTGACCATTGTCCC3′ (SEQ ID NO: 587).Complementarity between the two PCR products was used to drive annealingof the two fragments in a PCR assembly reaction and the full lengthrecombined scFv library was amplified with Reverse Tag GAC ACC TCG ATCAGC G (SEQ ID NO: 588) and HuJk 2-3 FOR NOT oligonucleotide 5′GAG TCATTC TCG ACT TGC GGC CGC ACC TAG GAC GGT CAG CTT GGT CCC 3′ (SEQ ID NO:589).

Heavy chain CDR3 mutants were selected using 1 nM biotinylated IL-12,and washed for 1 h at room temperature in PBS containing free IL-12 orp40 at a concentration of 7 nM. Clones were analyzed by phage ELISA andthose that bound to IL-12 were tested in BIAcore kinetic binding studiesusing a low density IL-12 chip (see procedure for BIAcore analysis inExample 5). Generally, BIAcore analysis measures real-time bindinginteractions between ligand (recombinant human IL-12 immobilized on abiosensor matrix) and analyte (antibodies in solution) by surfaceplasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor,Piscataway, N.J.). The system utilizes the optical properties of SPR todetect alterations in protein concentrations within a dextran biosensormatrix. Proteins are covalently bound to the dextran matrix at knownconcentrations. Antibodies are injected through the dextran matrix andspecific binding between injected antibodies and immobilized ligandresults in an increased matrix protein concentration and resultantchange in the SPR signal. These changes in SPR signal are recorded asresonance units (RU) and are displayed with respect to time along they-axis of a sensorgram. To determine the off rate (k_(off)), on rate(k_(on)), association rate (Ka) and dissociation rate (Kd) constants,BIAcore kinetic evaluation software (version 2.1) was used. Clones thatdemonstrated an improvement in the k_(off) rate were analyzed byneutralization assays which included inhibition by antibody of IL-12binding to its receptor (RBA assay), inhibition of IL-12-inducedproliferation in PHA stimulated human blast cells (PHA assay), andinhibition of IL-12-induced interferon gamma production by human blastcells (IFN gamma assay). A summary of the dissociation rates and/or IC₅₀values from neutralization assays of heavy chain CDR3 spiked clones 70-1through 70-13 is presented in Table 2 (see Appendix A). Clone 70-1displayed a k_(off) rate that was better than the parent Joe 9 clone,and had the lowest IC₅₀ value of 2.0×10⁻⁷ M. Therefore clone 70-1 wasselected for conversion to complete IgG1.

Light chain CDR3 mutants were selected using 1 nM biotin-IL-12 andwashed with PBS containing 7 nM free p40. Clones were screened in phageELISA and those that bound to IL-12 were tested in BIAcore bindinganalysis using low density IL-12 chips. Clones that displayed an offrate which was better than the parent Joe 9 clone were tested inneutralization assays which measured either, inhibition of IL-12receptor binding, or inhibition of PHA blast cell proliferation. Asummary of the dissociation rates and/or IC₅₀ values from neutralizationassays of light chain CDR3 mutant clones, 78-34 through 79-1, ispresented in Table 2 (see Appendix A).

Based on the k_(off) rate, clones 78-34 and 78-35 displayed an improvedk_(off) rate compared to the parent Joe 9. Both of these clones wereselected for combination analysis with heavy chain mutants.

C. Combination Clones

Mutant light and heavy chain clones that exhibited the best bindingcharacteristics were used for combination and assembly of scFvs. Mutantclones with improved potency characteristics were combined by PCRoverlap extension and pull-through of the mutated VH and VL segments asdescribed above. Clones 101-14 through 26-1, shown in Table 2 (seeAppendix A), were produced from the combination of heavy chain mutants(70-2, 70-13 and 70-1) with light chain mutants (78-34, 78-35 and 79-1).The k_(off) rates and/or IC₅₀ values from neutralization assays forthese clones are presented in Table 2.

BIAcore binding analysis identified clone 101-11, produced from thecombination of the heavy chain CDR3 mutant clone 70-1 with the lightchain CDR3 mutant clone 78-34, as having an off rate of 0.0045^(s)−1.This k_(off) rate was a significant improvement compared to the k_(off)rates for either the heavy chain CDR3 mutant clone 70-1 (0.0134^(s)−1),or for the light chain CDR3 mutant clone 78-34 (0.0164^(s)−1) alone.Furthermore, clone 101-11 showed a significant improvement inneutralization assays. Accordingly, clone 101-11 was selected foraffinity maturation as described below.

D. Affinity Maturation of Clone 101-11

Further affinity maturation of clone 101-11 consisted of repeat cyclesof PCR mutagenesis of both the heavy and light chain CDR3s of 101-11using spiked oligonucleotide primers. The clones were selected withdecreasing concentrations of biotinylated IL-12 (bio-IL-12). The bindingcharacteristics of the mutated clones was assessed by BIAcore bindinganalysis and RBA, PHA neutralization assays. The k_(off) rates and/orIC₅₀ values for clones 136-9 through 170-25 are presented in Table 2(see Appendix A). Clone 103-14 demonstrated an improved IC₅₀ value inboth the receptor binding assay and the PHA blast assay. Clone 103-14also demonstrated a low k_(off) rate, and accordingly was selected forfurther affinity maturation.

E. Generation and Selection of Randomized Libraries of Clone 103-14Light CDR3

The light chain CDR3 of clone 103-14 (QSYDRGFTGSMV (SEQ ID NO: 590)) wassystematically randomized in 3 segments using 3 different libraries asoutlined below, where X is encoded by a randomized codon of sequence NNSwith N being any nucleotide and S being either deoxycytosine ordeoxyguanidine.

L3.1 = XXXXXXFTGSMV (SEQ ID NO: 591) L3.2 = QSYXXXXXXSMV(SEQ ID NO: 592) L3.3 = QSYDRGXXXXXX (SEQ ID NO: 593)

Randomized mutagenesis of all three light chain CDRs (referred to asL3.1, L3.2, and L3.3) of clone 103-14 was performed. The heavy chainCDR3 (referred to as H3) of clone 103-14 was not mutated. Fourrandomized libraries based on clone 103-14 (H3 and L3.1, L3.2 & L3.3)were constructed and subjected to a large variety of selectionconditions that involved using limiting antigen concentration and thepresence or absence of excess free antigen (p40 and p70). The outputsfrom selections (clones 73-B1 through 99-G11) were screened primarily byBIAcore, and on occasion with RBA and are shown in Table 2 (see AppendixA).

Random mutagenesis of the light chain CDR of 103-14 generated clone Y61,which exhibited a significant improvement in IC₅₀ value compared to theparent clone 103-14. Y61 was selected for conversion to a whole IgG1.Whole Y61-IgG1 has an IC₅₀ value of approximately 130 pM determined bythe PHA assay. The IC₅₀ value was not affected by a 50 fold molar excessof free p40, demonstrating that free p40 did not cross-react with Y61anti-IL-12 antibody to thereby decrease the antibody binding to theheterodimer. The full length sequences of Y61 heavy chain variableregion and light chain variable region are shown below.

Y61 Heavy Chain Variable Region Peptide  Sequence                              CDR H1QVQLVESGGGVVQPGRSLRLSCAASFTFS SYGMH WVRQAPGKGLEWVA       CDR H2FIRYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCKT (SEQ ID NO: 23)CDR H3 HGSHDN WGQGTMVTVSS Y61 Light Chain Variable Region Peptide Sequence                            CDR L1QSVLTQPPSVSGAPGQRVTISC SGGRSNIGSNTVK WYQQLPGTAPKLLIY  CDR L2GNDQRPS GVPDRFSGSKSGTSASLAITGLQAEDEADYYC (SEQ ID NO: 24)    CDR L3QSYDRGTHPALL FGTGTKVTVLGCDR residues are assigned according to the Kabat definitions.

Example 2 Mutation of Y61 at Hypermutation and Contact Positions

Typically selection of recombinant antibodies with improved affinitiescan be carried out using phage display methods. This is accomplished byrandomly mutating combinations of CDR residues to generate largelibraries containing single-chain antibodies of different sequences.Typically, antibodies with improved affinities are selected based ontheir ability to reach an equilibrium in an antibody-antigen reaction.However, when Y61 scFV was expressed on phage surface and incubated withIL-12, selection conditions could not be found that would allow thesystem to reach normal antibody-antigen equilibrium. The scFV-phageremained bound to IL-12, presumably due to a non-specific interaction,since purified Y61 scFv exhibits normal dissociation kinetics. Since theusual methods of phage-display affinity maturation to Y61 (i.e. librarygeneration and selections by mutagenesis of multiple CDR residues) couldnot be utilized, a new strategy was developed in which individual CDRpositions were mutated.

This strategy involves selection of appropriate CDR positions formutation and is based on identification and selection of amino acidsthat are preferred selective mutagenesis positions, contact positions,and/or hypermutation positions. Contact positions are defined asresidues that have a high probability of contact with an antigen whenthe antigen interacts with the antibody, while hypermutation positionsare defined as residues considered to have a high probability forsomatic hypermutation during in vivo affinity maturation of theantibody. Preferred selective mutagenesis positions are CDR positionsthat are both contact and hypermutation positions. The Y61 antibody wasalready optimized in the CDR3 regions using the procedure described inExample 1, therefore it was difficult to further improve the area whichlies at the center of the antibody binding site using phage-displayselection methods. Greater improvements in activity were obtained bymutation of potential contact positions outside the CDR3 regions byeither removing a detrimental antigen-antibody contact or, engineering anew contact.

Amino acids residues of Y61 which were considered contact points withantigen, and those CDR positions which are sites of somatichypermutations during in vivo affinity maturation, are shown in Table 3(see Appendix A). For Y61 affinity maturation, 15 residues outside CDR3,3 residues within the L3 loop, and 5 residues in the H3 loop wereselected for PCR mutagenesis.

Y61 scFv gene was cloned into the pUC119(Sfi) plasmid vector formutagenesis. Oligonucleotides were designed and synthesized withrandomized codons to mutate each selected position. Following PCRmutagenesis, a small number of clones (˜24) were sequenced and expressedin a host cell, for example, in a bacterial, yeast or mammalian hostcell. The expressed antibody was purified and the k_(off) measured usingthe BIAcore system. Clones with improved off-rates, as compared to Y61,were then tested in neutralization assays. This procedure was repeatedfor other CDR positions. Individual mutations shown to have improvedneutralization activity were combined to generate an antibody with evengreater neutralization potency.

The Y61 CDR positions that were mutated in order to improveneutralization potency, and the respective amino-acid substitutions ateach position are shown in FIGS. 2A-2H. Off-rates, as determined byBIAcore analysis, are given. These off rates are also shown in thehistograms to the right of each table.

Results of these substitutions at positions H30, H32, H33, H50, H53,H54, H58, H95, H97, H101, L50, L92, L93, demonstrated that allamino-acid substitutions examined resulted in antibodies with pooreroff-rates than Y61. At positions H52, L32, and L50, only a one aminoacid substitution was found to improve the off-rate of Y61, all otherchanges adversely affected activity. For L50, this single Gly→Tyr changesignificantly (5-10 times) improved the neutralization potency of Y61.The results demonstrated the importance of these positions to Y61activity, and suggest that in most cases phage-display was able toselect for the optimal residues. However, at positions H31, H56, L30,and L94, several substitutions were found to improve Y61 off-rate,suggesting that these positions were also important for antigen binding,although the phage display approach did not allow selection of theoptimal residues.

Selective mutation of contact and hypermutation positions of Y61identified amino acid residue L50 in the light chain CDR2, and residueL94 of the light chain CDR3, which improved the neutralization abilityof Y61. A combination of these mutations produced an additive effect,generating an antibody, J695, that exhibited a significant increase inneutralization ability. The full length sequence of J695 heavy and lightchain variable region sequences is shown below.

J695 Heavy Chain Variable Region Peptide Sequence

                               CDR H1QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA     CDR H2FIRYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCKT (SEQ ID NO: 31)CDR H3 HGSHDN WGQGTMVTVSS J695 Light Chain Variable Region Peptide Sequence                           CDR L1QSVLTQPPSVSGAPGQRVTISC SGSRSNIGSNTVK WYQQLPGTAPKLLIY CDR L2YNDQRPS GVPDRFSGSKSGTSASLAITGLQAEDEADYYC (SEQ ID NO: 32)    CDR L3QSYDRYTHPALL FGTGTKVTVLGCDR residues are assigned according to the Kabat definitions.

A summary of the heavy and light chain variable region sequencealignments showing the lineage development of clones that were on thepath from Joe9 to J695 is shown in FIGS. 1A-1D. The CDRs and residuenumbering are according to Kabat.

Example 3 Functional Activity of Anti-hIL-12 Antibodies

To examine the functional activity of the human anti-human IL-12antibodies of the invention, the antibodies were used in several assaysthat measure the ability of an antibody to inhibit IL-12 activity.

A. Preparation of Human PHA-Activated Lymphoblasts

Human peripheral blood mononuclear cells (PBMC) were isolated from aleukopac collected from a healthy donor by Ficoll-Hypaque gradientcentrifugation for 45 minutes at 1500 rpm as described in CurrentProtocols in Immunology, Unit 7.1. PBMC at the interface of the aqueousblood solution and the lymphocyte separation medium were collected andwashed three times with phosphate-buffered saline (PBS) bycentrifugation for 15 minutes at 1500 rpm to remove Ficoll-Paqueparticles.

The PBMC were then activated to form lymphoblasts as described inCurrent Protocols in Immunology, Unit 6.16. The washed PBMC wereresuspended at 0.5-1×10⁶ cells/ml in RPMI complete medium (RPMI 1640medium, 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/mlstreptomycin), supplemented with 0.2% (v/v) PHA-P (Difco, Detroit,Mich.) and cultured for four days at 37° C. in a 5% CO₂ atmosphere.After four days, cell cultures were split 1:1 by volume in RPMI completemedium, plus 0.2% (v/v) PHA-P and 50 U/ml recombinant human IL-2.Recombinant human IL-2 was produced by transfection of an expressionvector carrying the human IL-2 cDNA into COS cells (see Kaufman et al.,(1991) Nucleic Acids Res. 19, 4484-4490), and purified as described inPCT/US96/01382. Cell cultures were then incubated for an additional oneto three days. PHA blast cells were harvested, washed twice with RPMIcomplete medium and frozen in 95% FBS, 5% DMSO at 10×10⁶ cells/ml.

PHA blast cells to be used for the IL-12 receptor binding assay (seesection B) were collected after one day culture in the presence of IL-2,whereas PHA blast cells to be used for the PHA blast proliferation assay(see section C) and the interferon-gamma induction assay (see section D)were collected after three day culture in the presence of IL-2.

B. IL-12 Receptor Binding Assay

The ability of anti-IL-12 antibodies to inhibit binding of radiolabelledIL-12 to IL-12 receptors on PHA blasts were analyzed as follows. Variousconcentrations of anti-IL-12 antibody were preincubated for 1 hour at37° C. with 50-100 pM ¹²⁵I-hIL-12 (iodinated hIL-12 was prepared usingthe Bolton-Hunter labeling method to a specific activity of 20-40 mCi/mgfrom NEN-Dupont) in binding buffer (RPMI 1640, 5% FBS, 25 mM Hepes pH7.4). PHA blast cells isolated as described above, were washed once andresuspended in binding buffer to a cell density of 2×10⁷ cells/ml. PHAblasts (1×10⁶ cells) were added to the antibody ¹²⁵I-hIL-12 mixture andincubated for two hours at room temperature. Cell bound radioactivitywas separated from free ¹²⁵I-hIL-12 by centrifugation of the assaymixture for 30 seconds at room temperature, aspiration of the liquid anda wash with 0.1 ml binding buffer, followed by centrifugation at 4° C.for 4 min at 10,000×g. The cell pellet was examined for cell boundradioactivity using a gamma counter. Total binding was determined in theabsence of antibody and non-specific binding was determined by inclusionof 25 nM unlabeled IL-12 in the assay. Incubations were carried out induplicate.

In the IL-12 receptor binding assay using the Y61 and J695 humananti-IL-12 antibodies, both antibodies demonstrated a comparableinhibition of IL-12 receptor binding. Y61 inhibited IL-12 receptorbinding with an IC₅₀ value of approximately 1.6×10⁻¹¹ M, while J695 hadan IC₅₀ value of approximately 1.1×10⁻¹¹ M.

C. Human PHA Blast Proliferation Assay

Anti-IL-12 antibodies were evaluated for their ability to inhibit PHAblast proliferation (which proliferation is stimulated by IL-12). Serialdilutions of anti-IL-12 antibody were preincubated for 1 hour at 37° C.,5% CO₂ with 230 pg/ml hIL-12 in 100 ml RPMI complete medium in amicrotiter plate (U-bottom, 96-well, Costar, Cambridge, Mass.). PHAblast cells isolated as described above, were washed once andresuspended in RPMI complete medium to a cell density of 3×10⁵ cells/ml.PHA blasts (100 ml, 3×10⁴ cells) were added to the antibody/hIL-12mixture, incubated for 3 days at 37° C., 5% CO₂ and labeled for 4-6hours with 0.5 mCi/well (3H)-Thymidine (Amersham, Arlington Heights,Ill.). The culture contents were harvested onto glass fiber filters bymeans of a cell harvester (Tomtec, Orange, Conn.) and (³H)-Thymidineincorporation into cellular DNA was measured by liquid scintillationcounting. All samples were assayed in duplicate.

The results of neutralization in the presence of varying concentrationsof p70:p40 (i.e. the ratio of IL-12 heterodimer to free p40 subunit) isshown in Table 4 (see Appendix A).

Analysis of the Y61 human anti-IL-12 antibody in the PHA blastproliferation assay demonstrated that the antibody inhibited PHA blastproliferation with an IC₅₀ value of approximately 1.8×10⁻¹⁰ M in thepresence of IL-12 p70 alone, without any excess p40 (p70:p40 ratio of1:0). In the presence of a 50-fold excess of free p40 (p70:p40 at aratio of 1:50), the Y61 antibody inhibited PHA blast proliferation withan IC₅₀ value of approximately 1.8×10⁻¹⁰ M. This result demonstratesthat the ability of Y61 to inhibit blast proliferation is notcompromised by the presence of excess p40.

The human anti-IL-12 antibody, J695 inhibited PHA blast proliferationwith an IC₅₀ value of approximately 1.0×10⁻¹¹ M in the presence ofp70:p40 at a ratio of 1:0. In the presence of a p70:p40 ratio of 1:50,this antibody inhibited PHA blast proliferation with an IC₅₀ value ofapproximately 5.8±2.8×10⁻¹² M (n=2), demonstrating that the excess p40had only a slight inhibitory effect on the antibody. Overall resultsdemonstrate the improved neutralization activity of J695 in comparisonwith Y61 due to the mutations at L50 and L94.

D. Interferon-Gamma Induction Assay

The ability of anti-IL-12 antibodies to inhibit the production of IFNγby PHA blasts (which production is stimulated by IL-12) was analyzed asfollows. Various concentrations of anti-IL-12 antibody were preincubatedfor 1 hour at 37° C., 5% CO₂ with 200-400 pg/ml hIL-12 in 100 ml RPMIcomplete medium in a microtiter plate (U-bottom, 96-well, Costar). PHAblast cells isolated as described above, were washed once andresuspended in RPMI complete medium to a cell density of 1×10⁷ cells/ml.PHA blasts (100 μl of 1×10⁶ cells) were added to the antibody/hIL-12mixture and incubated for 18 hours at 37° C. and 5% CO₂. Afterincubation, 150 μl of cell free supernatant was withdrawn from each welland the level of human IFNγ produced was measured by ELISA (EndogenInterferon gamma ELISA, Endogen, Cambridge, Mass.). Each supernatant wasassayed in duplicate.

Analysis of human anti-hIL-12 antibody, Y61 in this assay demonstratedthat Y61 inhibited human IFNγ production with an IC₅₀ value ofapproximately 1.6×10⁻¹⁰ M, while the human anti-IL-12 antibody, J695,inhibited human IFNγ production with an IC₅₀ value of approximately5.0±2.3×10⁻¹² M (n=3). The result demonstrates the substantialimprovement in the affinity of J695 as a result of the modifications atL50 and L94.

E. Induction of Non-Human IL-12 from Isolated PBMC

To examine the cross-reactivity of the human anti-hIL-12 antibodies withIL-12 from other species, non-human IL-12 was produced as follows. PBMCwere separated from fresh heparinized blood by density gradientcentrifugation as described above using lymphoprep (Nycomed, Oslo,Norway) for cynomolgus monkey, baboon, and dog, PBMC, Accu-paque(Accurate Chemical & Sci. Corp., Westbury, N.Y.) for dog PBMC orLympholyte-rat (Accurate Chemical & Sci. Corp., Westbury, N.Y.) for ratPBMC.

The PBMC were then induced to produce IL-12 as described (D'Andrea etal., (1992) J. Exp. Med 176, 1387-1398, Villinger et al., (1995) J.Immunol. 155, 3946-3954, Buettner et al., (1998) Cytokine 10, 241-248).The washed PBMC were resuspended at 1×¹06 cells/ml in RPMI completemedium, supplemented with 0.0075% (wt/vol) of SAC (Pansorbin;Calbiochem-Behring Co., La Jolla, Calif.) or 1-5 mg/ml ConA (SigmaChemical Co., St. Louis, Mo.) plus 0.0075% SAC and incubated for 18hours at 37° C. in a 5% CO₂ atmosphere. Cell-free and SAC-free mediumwas collected by centrifugation and filtering through 0.2 mm filters.

IL-12 from the rhesus monkey was obtained as recombinant rhesus IL-12from Emory University School of Medicine, Atlanta, Ga.

F. Murine 2D6 Cell Proliferation Assay

The murine T cell clone 2D6 proliferates in response to murine IL-2,IL-4, IL-7 and IL-12 (Maruo et al., (1997) J. Leukocyte Biol. 61,346-352). A significant proliferation was also detected in response torat PBMC supernatants containing rat IL-12. The cells do not respond todog, cynomolgus, baboon or human IL-12. Murine 2D6 cells were propagatedin RPMI complete medium supplemented with 50 mM beta-mercaptoethanol(βME) and 30 ng/ml murine IL-12. One day prior to the assay, the murineIL-12 was washed out and the cells were incubated overnight in RPMIcomplete medium plus βME.

Serial dilutions of anti-IL-12 antibody were preincubated for 1 hour at37° C., 5% CO₂ with 40 pg/ml murine IL-12 in 100 ml RPMI complete mediumplus βME in a microtiter plate (U-bottom, 96-well, Costar). 2D6 cellswere washed once and resuspended in RPMI complete medium containinγβMEto a cell density of 1×10⁵ cells/ml. 2D6 cells (100 μl, 1×10⁴ cells)were added to the antibody/hIL-12 mixture, incubated for 3 days at 37°C., 5% CO₂ and labeled for 4-6 hours with 0.5 mCi/well (3H)-Thymidine.The culture contents were harvested and counted by liquid scintillationcounting. All samples were assayed in duplicate.

G. Species Cross-Reactivity of J695 with Non-Human IL-12

Species cross-reactivity of J695 with non-human IL-12 was analyzed usingPBMC's isolated from several non-human species. The presence ofnon-human IL-12 activity in the rat, dog, cynomolgus and baboon PBMCsupernatants was confirmed using several bioassays described above, suchas the murine 2D6 cell proliferation assay, the human PHA blastproliferation assay and the interferon-gamma induction assay by blockingthe non-human PBMC induced responses with rabbit and/or sheep polyclonalantibodies to murine and/or human IL-12. Cross-reactivity of the humananti-hIL-12 antibodies Y61 and J695 with non-human IL-12 in PBMCsupernatants or purified murine and rhesus IL-12 was then assessed inthe same bioassay(s) by determining the J695 antibody concentration atwhich 50% inhibition of the response was observed. The speciescross-reactivity results are summarized in Table 5. The resultsdemonstrate that Y61 and J695 are each able to recognize IL-12 frommonkeys (e.g., cynomolgus and rhesus IL-12 for Y61, and cynomolgus,rhesus and baboon for J695) and that J695 is approximately 35 fold lessactive on dog IL-12; neither Y61 nor J695 cross reacts with mouse or ratIL-12.

H. Human Cytokine Specificity of J695

The specificity of J695 was tested in a competition ELISA in which apanel of human cytokines was tested for their ability to interfere withthe binding of soluble J695 to immobilized human IL-12. The panel ofhuman cytokines included IL-1α and IL-1β (Genzyme, Boston, Mass.), IL-2(Endogen), IL-4, IL-10, IL-17, IFN-gamma, and TGF-β1 (R&D, Minneapolis,Minn.) IL-8 (Calbiochem), PDGF, IGF-I, and IGF-II (Boehringer MannheimCorp., Indianapolis, Ind.), TNFα and lymphotoxin, IL-6, soluble IL-6receptor, IL-11, IL-12 p70, IL-12 p40, M-CSF, and LIF. EBI-3, an IL-12p40 related protein that is induced by Epstein-Barr virus infection in Blymphocytes (Devergne et

TABLE 5 Species Cross Reactivity Data IC₅₀ (M) Antibody Mouse IL-12 RatIL-12 Dog IL-12 Cyno IL-12 Rhesus IL-12 Baboon IL-12 Human IL-12 NameSpecificity Purified PBMC sup PBMC sup PBMC sup Purified PBMC supPurified C17.15 rat-αmuIL12 3.0 × 10⁻¹¹ R03B03 rabbit-αmuIL12 1.5 ×10⁻¹⁰ 6.0 × 10⁻¹⁰ C8.6.2 mouse-αhuIL12 1.2 × 10⁻¹⁰ 1.0 × 10⁻¹⁰ 2.0 ×10⁻¹⁰ 5.0 × 10⁻¹¹ Y61 human-αhuIL12 Non-neutralizing 2.2 × 10⁻¹⁰ 1.0 ×10⁻¹⁰ 1.7 × 10⁻¹⁰ J695 human-αhuIL12 Non-neutralizing Non-neutralizing3.5 × 10⁻¹⁰ 1.0 × 10⁻¹¹ 1.0 × 10⁻¹¹ 1.5 × 10⁻¹¹ 5.0 × 10⁻¹²al., (1996) J. Virol. 70, 1143-1153) was expressed as a human IgG-Fcchimera (EBI-3/Fc) Single-stranded salmon sperm DNA (Sigma) was alsotested.

Flat-bottom ELISA immunoassay microtiter plates (96 well, high binding,Costar) were coated overnight at 4° C. with 0.1 ml human IL-12 (2 μg/mlin 0.1 M carbonate coating buffer (4 volumes 0.1 M NaHCO₃ plus 8.5volumes 0.1 M NaHCO₃)). The plates were washed twice with PBS containing0.05% Tween 20 (PBS-T), blocked with 200 μl of 1 mg/ml bovine serumalbumin (BSA, Sigma) in PBS-T for 1 hour at room temperature, and againwashed twice with PBS-T. Samples (100 μl) containing IL-12 antibody J695(100 ng/ml) and each cytokine (2 nM) in PBS-T containing 50 μg/ml BSA(PBS-T/BSA) were added and incubated for 2 h at room temperature. Theplates were washed 4 times and incubated for 1 h at room temperaturewith 100 μl mouse anti-human lambda-HRP (1:500 in PBS-T/BSA, SouthernBiotech. Ass. Inc., Birmingham, Ala.). The plates were washed 4 timesand developed with ABTS (Kirkegaard & Perry Lab, Gaithersburg, Md.) for20-30 minutes in the dark. The OD₄₅₀ nm was read using a microplatereader (Molecular Devices, Menlo Park, Calif.). Percent binding wasdetermined relative to J695 binding to the IL-12 coated plate in theabsence of any soluble cytokine.

The results demonstrated that J695 binding to immobilized human IL-12was blocked only by human IL-12 p70 and to a lesser extent, by humanIL-12 p40 and not by any of the other cytokines tested.

I. Binding to a Novel IL-12 Molecule

An alternative IL-12 heterodimer has been described, in which the p35subunit is replaced by a novel p19 molecule. P19 was identified using 3Dhomology searching for IL-6/IL-12 family members, and is synthesized byactivated dendritic cells. P19 binds to p40 to form a p19/p40 dimer,which has IL-12-like activity, but is not as potent as the p35/p40heterodimer in IFNγ induction. Antibodies which recognize p40 alone, butpreferably in the context of a p70 molecule (e.g., J695 and Y61, seeExample 3H) are expected to also neutralize both the p35/p40 moleculesand the p19/p40 molecules.

Example 4 In Vivo Activity of Anti-hIL-12 Antibodies

The in vivo effects of IL-12 antibodies on IL-12 induced responses wereexamined in a model modified from one used by Bree et al. to study theeffect of human IL-12 on peripheral hematology in cynomolgus monkey Breeet al., (1994) Biochem Biophys Res. Comm. 204: 1150-1157. In thoseprevious studies, administration of human IL-12 at 1 μg/kg/day for aperiod of 5 days resulted in a decrease in white blood cell count (WBC),especially in the lymphocyte and monocyte subsets after 24 hours. Adecrease in the platelet count was observed at 72 hours. Levels ofplasma neopterin, a marker of monocyte activation in response to IFN-γ,began to elevate at 24 hours and were the highest at 72 hours.

In the first study with human anti-hIL-12 antibodies, fifteen healthycynomolgus monkeys with an average weight of 5 kg, were sedated anddivided into 5 groups (n=3). Group 1 received an intravenous (IV)administration of 10 mg/kg human intravenous immunoglobulin (IVIG,Miles, Eckhart, Ind., purified using protein A Sepharose). Group 2received an intravenous administration of 1 mg/kg C8.6.2 (neutralizingmouse anti-human IL-12 monoclonal antibody). Group 3 received anintravenous administration of 10 mg/kg C8.6.2. Group 4 received anintravenous administration of 1 mg/kg Y61 (human anti-human IL-12antibody, purified from CHO cell conditioned medium). Group 5 receivedan intravenous administration of 10 mg/kg Y61.

One hour after the antibody administration all animals received a singlesubcutaneous (SC) injection of human IL-12 (1 μg/kg). Blood samples weretaken at the following time points: baseline, 8, 24, 48, 96 and 216hours, and analyzed for complete blood cell counts with differentialsand serum chemistry. Serum human IL-12, C8.6.2 antibody, Y61 antibody,monkey IFN-gamma, monkey IL-10, monkey IL-6 and plasma neopterin levelswere also measured.

Animals treated with IL-12 plus IVIG control antibody (Group 1) showedmany of the expected hematological changes, including decreases in WBC,platelets, lymphocyte count and monocyte count. These decreases were notseen or were less pronounced in the animals treated with either theC8.6.2 or Y61 antibody at 1 or 10 mg/kg (Groups 2-5).

Serum or plasma samples were analyzed by ELISA specific for monkeyIFN-gamma and monkey IL-10 (Biosource International, Camarillo, Calif.),monkey IL-6 (Endogen) and plasma neopterin (ICN Pharmaceuticals,Orangeburg, N.Y.). IFN-gamma, IL-10 or IL-6 were not detected in any ofthe IL-12 treated animals including the control animals treated withIL-12 plus IVIG. This was probably due to the low level exposure toIL-12 (only 1 dose of 1 μg/kg). Nevertheless, plasma neopterin levelsincreased about three fold in the IL-12 plus IVIG treated animals butdid not change in all C8.6.2 or Y61 treated animals, including the lowerdose (1 mg/kg) Y61 treated animals, indicating that Y61 was effective invivo in blocking this sensitive response to IL-12.

In a second study, in vivo activity and pharmacodynamics (PD) of J695 incynomolgous monkeys were studied by administering exogenous rhIL-12 anddetermining if J695 could block or reduce the responses normallyassociated with rhIL-12 administration. Male cynomolgus monkeys (n=3 pergroup) were administered a single dose of 0.05, 0.2, or 1.0 mg/kg J695or 1 mg/kg intravenous immunoglobulin (IVIG) as a bolus intravenous (IV)injection via a saphenous vein or subcutaneously (SC) in the dorsalskin. One hour following the administration of J695 or IVIG, all animalsreceived a single SC dose of 1 μg/kg rhIL-12 in the dorsal skin. Bloodsamples were collected via the femoral vein up to 28 days after J695administration. Serum was acquired from each blood sample and assayedfor IL-12, J695, IFN-γ, and anti-J695 antibodies by ELISA. Neopterin wasassayed by reverse-phase high performance liquid chromatography.

The levels of neopterin, normalized with respect to the levels ofneopterin that were measured before administration of J695 or rhIL-12,are shown in FIG. 3. To compare the suppression of neopterin betweengroups, the area under the curve (AUC) normalized for neopterin levelswas calculated for each animal (Table 6). Neopterin exposure (AUC) wassuppressed in a dose-dependent manner between approximately 71 and 93%in the IV groups and between 71 and 100% in SC groups, relative to theIVIG control groups. These results suggest that the dose of J695necessary for 50% inhibition of the neopterin response (ED₅₀) was lessthan 0.05 mg/kg when administered by either the IV or SC route.

TABLE 6 Dose-Dependent Suppression of IL-12 Induced Neopterin by J695 inCynomolgus Monkeys AUC of % Reduction of J695 IVIG Normalized NeopterinAUC Route of dosing IVIG Dose Dose Neopterin Compared with or J695 andrhIL-12 (mg/kg) (mg/kg) Levels Control Single IV injection — 1.0 1745 ±845  0 followed 1 hr later  0.05 — 502 ± 135 71.3 by a dose of 1 μg/kg0.2 — 199 ± 316 88.6 human IL-12 given SC 1.0 — 128 ± 292 92.7 Single SCinjection — 1.0 1480 ± 604  0 followed 1 hour later  0.05 — 426 ± 10871.2 by a dose of 1 μg/kg 0.2 —  395 ± 45.9 73.3 human IL-12 given SC1.0 —  0 ± 109 100

Treatment with J695 also prevented or reduced the changes in hematologynormally associated with rhIL-12 administration (leukopenia andthrombocytopenia). At 24 hours after rhIL-12 administration lymphocytecounts were reduced by approximately 50% when compared to baselinevalues in the control IV and SC IVIG treated groups. Administration ofJ695 either SC or IV at all three dose levels prevented this reduction,resulting in lymphocyte counts at 24 hours approximately the same asbaseline values. At 48 hours after IL-12 administration, platelet countsin the groups treated with IV and SC IVIG were reduced by approximately25% when compared to baseline values.

An example dose schedule targeted to maintain serum levels above the 90%effect level would be 1 mg/kg IV and SC given approximately every otherweek, or 0.3 mg/kg given approximately every week, assuming slightaccumulation during repeated dosing. This study demonstrates thatantibody can be given safely to monkeys at such dosages. In independenttoxicity studies, it was further found that up to 100 mg/kg of theantibody can be given safely to monkeys.

J695 was also effective in preventing IFN-γ production in mice treatedwith a chimeric IL-12, a molecule which combines the murine p35 subunitwith the human IL-12 p40 subunit. In contrast to human IL-12 which isbiologically inactive in mice, this chimeric IL-12 retains biologicalfunction in mice, including induction of IFN-γ. In addition, the humanp40 subunit allows the molecule to be bound and neutralized by J695.Chimeric IL-12 at a dose of 0.05 mg/kg i.p. was administered to femaleC3H/HeJ mice (10/experimental group) in five daily doses on days 0, 1,2, 3, and 4. J695 was given on days 0, 2 and 4 at doses of 0.05, 0.01,0.002, 0.0004, 0.00008, and 0.000016 mg/kg i.p., 30′ prior to the IL-12injections. The control hulgG1γ was given IP. at a dose of 0.05 mg/kg ondays 0, 2, and 4. The mice were bled on day 5, and serum IFN-γ levelswere determined by ELISA. The results demonstrated that J695 causeddose-dependent inhibition of IFN-γ production with an ED₅₀ ofapproximately 0.001 mg/kg. Collectively, these results demonstrate thatJ695 is a potent inhibitor of 11-12 activity in vivo.

Example 5 Kinetic Analysis of Binding of Human Antibodies to RecombinantHuman IL-12 (rhIL-12)

Real-time binding interactions between captured ligand (humananti-rhIL-12 antibody J695, captured on a biosensor matrix) and analyte(rhIL12 in solution) were measured by surface plasmon resonance (SPR)using the BIAcore system (Biacore AB, Uppsala, Sweden). The systemutilizes the optical properties of SPR to detect alterations in proteinconcentration within a dextran biosensor matrix. Proteins are covalentlybound to the dextran matrix at known concentrations. Antibodies areinjected through the dextran matrix and specific binding betweeninjected antibodies and immobilized ligand results in an increasedmatrix protein concentration and resultant change in the SPR signal.These changes in SPR signal are recorded as resonance units (RU) and aredisplayed with respect to time along the y-axis of a sensorgram.

To facilitate immobilization of goat anti-human IgG (SouthernBiotechnology Associates, Cat. No. 2040-01, Birmingham, Ala.) on thebiosensor matrix, goat anti-human IgG is covalently linked via freeamine groups to the dextran matrix by first activating carboxyl groupson the matrix with 100 mM N-hydroxysuccinimide (NHS) and 400 mMN-Ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC).Next, goat anti-human IgG is injected across the activated matrix.Thirty-five microliters of goat anti-human IgG (25 μg/ml), diluted insodium acetate, pH 4.5, is injected across the activated biosensor andfree amines on the protein are bound directly to the activated carboxylgroups. Unreacted matrix EDC-esters are deactivated by an injection of 1M ethanolamine. Standard amine coupling kits were commercially available(Biacore AB, Cat. No. BR-1000-50, Uppsala, Sweden).

J695 was diluted in HBS running buffer (Biacore AB, Cat. No. BR-1001-88,Uppsala, Sweden) to be captured on the matrix via goat anti-human IgG.To determine the capacity of rhIL12-specific antibodies to bindimmobilized goat anti-human IgG, a binding assay was conducted asfollows. Aliquots of J695 (25 μg/ml; 25 μl aliquots) were injectedthrough the goat anti-human IgG polyclonal antibody coupled dextranmatrix at a flow rate of 5 μl/min. Before injection of the protein andimmediately afterward, HBS buffer alone flowed through each flow cell.The net difference in signal between the baseline and the pointcorresponding to approximately 30 seconds after completion of J695injection was taken to represent the amount of IgG1 J695 bound(approximately 1200 RU's). Direct rhIL12 specific antibody binding tosoluble rhIL12 was measured. Cytokines were diluted in HBS runningbuffer and 50 μl aliquots were injected through the immobilized proteinmatrices at a flow rate of 5 μl/min. The concentrations of rhIL-12employed were 10, 20, 25, 40, 50, 80, 100, 150 and 200 nM. Prior toinjection of rhIL-12, and immediately afterwards, HBS buffer aloneflowed through each flow cell. The net difference in baseline signal andsignal after completion of cytokine injection was taken to represent thebinding value of the particular sample. Biosensor matrices wereregenerated using 100 mM HCl before injection of the next sample. Todetermine the dissociation constant (off-rate), association constant(on-rate), BIAcore kinetic evaluation software (version 2.1) was used.

Representative results of CHO derived J695 binding to rhIL-12 ascompared to the COS derived J695, are shown in Table 7.

TABLE 7 Binding of CHO or COS derived J695 to rhIL-12. rhIL12, rhIL12bound, Ab, bound, Source nM RU's RU's rhIL12/AB CHO 200 1112 1613 1.48CHO 150 1033 1525 1.45 CHO 100 994 1490 1.43 CHO 80 955 1457 1.40 CHO 50912 1434 1.36 CHO 40 877 1413 1.33 CHO 25 818 1398 1.25 CHO 20 773 13821.20 CHO 10 627 1371 0.98 COS 200 1172 1690 1.49 COS 150 1084 1586 1.46COS 100 1024 1524 1.44 COS 80 985 1489 1.42 COS 50 932 1457 1.37 COS 40894 1431 1.34 COS 25 833 1409 1.27 COS 20 783 1394 1.20 COS 10 642 13771.00

Molecular kinetic interactions between captured J695 and soluble rhIL-12were quantitatively analyzed using BIAcore technology. Severalindependent experiments were performed and the results were analyzed bythe available BIAcore mathematical analysis software to derive kineticrate constants, as shown in Table 8.

TABLE 8 Apparent kinetic rate and affinity constants of J695 forrhIL-12. On-rate (M−1s−1), Off-rate (s−1), Kd (M), Antibody Source Avg.Avg. Avg. J695 CHO 3.52E+05 4.72E−05 1.34E−10 J695 COS 3.40E+05 2.61E−059.74E−11

There was a small difference between the calculated apparent constant(Kd) for the interaction between CHO derived J695 (Kd=1.34⁻¹⁰ M⁻¹) andCOS derived J695 (Kd=9.74×10⁻¹¹ M⁻¹) antibodies. The apparentdissociation constant (Kd) between J695 and rhIL12 was estimated fromthe observed rate constants by the formula: Kd=off-rate/on-rate.

To determine the apparent association and dissociation rate constant forthe interaction between J695 and rhIL-12, several binding reactions wereperformed using a fixed amount of J695 (2 μg/ml) and varyingconcentrations of rhIL-12. Real-time binding interaction sensorgramsbetween captured J695 and soluble rhIL12 showed that both forms ofantibody were very similar for both the association and dissociationphase.

To further evaluate the capacity of captured IgG1 J695 mAb to bindsoluble recombinant cytokine, a direct BIAcore method was used. In thismethod, goat anti-human IgG (25 μg/ml) coupled carboxymethyl dextransensor surface was coated with IgG1 J695 (2 μg/ml) and recombinantcytokine was then added. When soluble rhIL12 was injected across abiosensor surface captured with CHO or COS derived IgG1 J695, the amountof signal increased as the concentration of cytokine in the solutionincreased. No binding was observed with rmIL12 (R&D Systems, Cat. No.419-ML, Minneapolis, Minn.) or rh IL12 any concentration tested up to1000 nM. These results support the conclusion that IgG1 J695 antibodiesrecognize a distinct determinant on rhIL-12.

Table 9 shows the results of an experiment using BIAcore to demonstratehuman IgG1 J695 mAb binding to only soluble rhIL12 and none of the otherrecombinant cytokines.

TABLE 9 Epitope mapping of J695 using BIAcore technology. Capturedligand Captured ligand Soluble analyte COS J695 CHO J695 rec. human IL12Positive Positive rec. murine IL12 Negative Negative

Example 6 Further Studies of J695 Affinity for IL-12

Molecular kinetic interactions between J695 antibody and human IL-12were quantitatively analyzed using BIAcore plasmon resonance technology,and apparent kinetic rate constants were derived.

BIAcore technology was used to measure the binding of soluble rhIL-12 tosolid phase captured J695. A goat anti-human IgG antibody wasimmobilized on the biosensor chips, then a fixed amount of J695 wasinjected and captured on the surface. Varying concentrations of rhIL-12were applied, and the binding of IL-12 at different concentrations toJ695 was measured as a function of time. Apparent dissociation andassociation rate constants were calculated, assuming zero-orderdissociation and first order association kinetics, as well as a simpleone-to-one molecular interaction between J695 and IL-12. Threeindependent experiments were performed, and the values shown areaverages for the three experiments. From these measurements, theapparent dissociation (k_(d)) and association (k_(a)) rate constantswere derived and used to calculate a K_(d) value for the interaction(see Table 10). The results indicated that J695 has a high affinity forrhIL-12.

TABLE 10 Kinetic Parameters for the Interaction Between J695 and HumanIL-12 Kinetic Parameter Value k_(d) 3.71 ± 0.40 × 10⁻⁵ s⁻¹ k_(a) 3.81 ±0.48 × 10⁵ M⁻¹s⁻¹ K_(d) 9.74 × 10⁻¹¹ M (14 ng/mL)

Example 7 Characteristics and Neutralization Activity of C17.15, a RatMonoclonal Antibody to Murine Interleukin-12

To assess the relevance of IL-12 treatment studies in mouse models ofinflammation and autoimmunity using monoclonal antibodies specific formurine IL-12 to similar approaches in human disease, the interaction ofC17.15, a rat anti-murine IL-12 monoclonal antibody with murine IL-12,was examined. The ability of C17.15 to neutralize murine IL-12 activityin a PHA blast proliferation assay, and to block murine IL-12 binding tocell surface receptors, was assessed, as were the kinetics of theC17.15-murine IL-12 binding interaction.

In a human PHA blast proliferation assay (See Example 3), serialdilutions of C17.15 or rat IgG2a (a control antibody) were preincubatedwith 230 pg/mL murine IL-12 for 1 hr at 37° C. PHA-stimulated blastcells were added to the antibody-IL-12 mixtures and incubated for 3 daysat 37° C. The cells were subsequently labeled for 6 h with 1 μCi/well[³H]-thymidine. The cultures were harvested and [³H]-thymidineincorporation was measured. Background non-specific proliferation wasmeasured in the absence of added murine IL-12. All samples were assayedin duplicate. The IC₅₀ (M) of C17.15 for recombinant murine IL-12 inthis assay was found to be 1.4×10⁴¹, as compared to the IC₅₀ value of5.8×10⁻¹² observed for J695 for recombinant human IL-12 under the sameconditions (see Table 11).

TABLE 11 Comparison of the properties of anti-human IL-12 monoclonalantibody J695 and the rat anti-mouse IL-12 monoclonal antibody C17.15Biomolecular Interaction Assay Receptor k_(a), on-rate k_(d), off-rateBinding PHA blast Antibody Epitope (M⁻¹s⁻¹) (s⁻¹) K_(d) (M) IC₅₀ (M)IC₅₀ (M) J695 Hu p40 3.81 × 10⁵ 3.71 × 10⁻⁵ 9.74 × 10⁻¹¹ 1.1 × 10⁻¹¹ 5.8× 10⁻¹² C17.15 Mu p40 3.80 × 10⁵ 1.84 × 10⁻⁴ 4.80 × 10⁻¹⁰ 1.5 × 10⁻¹⁰1.4 × 10⁻¹¹

The ability of C17.15 to inhibit the binding of murine IL-12 to cellularreceptors was also measured. Serial dilutions of C17.15 werepre-incubated for 1 hr at 37° C. with 100 pM [¹²⁵I]-murine IL-12 inbinding buffer. The 2D6 cells (2×10⁶) were added to theantibody/[¹²⁵I]-murine IL-12 mixture and incubated for 2 hours at roomtemperature. Cell-bound radioactivity was separated from free[¹²⁵I]-IL-12, and the remaining cell-bound radioactivity was determined.Total binding of the labeled murine IL-12 to receptors on 2D6 cells wasdetermined in the absence of antibody, and non-specific binding wasdetermined by the inclusion of 25 nM unlabelled murine IL-12 in theassay. Specific binding was calculated as the total binding minus thenon-specific binding. Incubations were carried out in duplicate. Theresults showed that C17.15 has an IC₅₀ (M) of 1.5×10⁻¹⁰ for inhibitionof binding of murine IL-12 to cellular receptors.

The affinity of C17.15 for recombinant murine IL-12 was assessed bybiomolecular interaction analysis. A goat anti-rat IgG antibody wasimmobilized on the biosensor chips, followed by an injection of a fixedamount of the C17.15 antibody, resulting in capture of C17.15 on thesurface of the chip. Varying concentrations of recombinant murine IL-12were applied to the C17.15 surface, and the binding of murine IL-12 tothe immobilized C17.15 was measured as a function of time. Apparentdissociation and association rate constants were calculated, assuming azero order dissociation and first order association kinetics as well asa simple one to one molecular interaction between the immobilized C17.15and murine IL-12. From these measurements, the apparent dissociation(k_(d), off-rate) and association (k_(a), on-rate) rate constants werecalculated. These results were used to calculate a K_(d) value for theinteraction. An on-rate of 3.8×10⁵ M⁻¹s⁻¹, an off-rate of 1.84×10⁻⁴ s⁻¹,and a K_(d) of 4.8×10⁻¹⁰ was observed for the recombinant murineIL-12-C17.15 interaction.

The observed activities of C17.15 in neutralizing murine IL-12 activityand binding to cell surface receptors, as well as the kinetics ofbinding of C17.15 to murine IL-12 correlate with similar measurementsfor the J695-rhIL-12 interaction. This indicates that the modes ofaction of the rat anti-mouse IL-12 antibody C17.15 and anti-human IL-12antibody J695 are nearly identical based upon on-rate, off-rate, K_(d),IC₅₀, and the PHA blast assay. Therefore, C17.15 was used as ahomologous antibody to J695 in murine models of inflammation andautoimmune disease to study the effects of IL-12 blockade on theinitiation or progression of disease in these model animals (see Example8).

Example 8 Treatment of Autoimmune or Inflammation-Based Diseases in Miceby α-Murine IL-12 Antibody Administration

a. Suppression of Collagen-Induced Arthritis in Mice by the α-II-12Antibody C17.15

A correlation between IL-12 levels and rheumatoid arthritis (RA) hasbeen demonstrated. For example, elevated levels of IL-12 p70 have beendetected in the synovia of RA patients compared with healthy controls(Morita et al (1998) Arthritis and Rheumatism. 41: 306-314). Therefore,the ability of C17.15, a rat anti-mouse IL-12 antibody, to suppresscollagen-induced arthritis in mice was assessed.

Male DBA/1 mice (10/group) were immunized with type II collagen on Day 0and treated with C17.15, or control rat IgG, at 10 mg/kgintraperitoneally on alternate days from Day −1 (1 day prior to collagenimmunization) to Day 12. The animals were monitored clinically for thedevelopment of arthritis in the paws until Day 90. The arthritis wasgraded as: 0—normal; 1—arthritis localized to one joint; 2—more than onejoint involved but not whole paw; 3—whole paw involved; 4—deformity ofpaw; 5—ankylosis of involved joints. The arthritis score of a mouse wasthe sum of the arthritic grades in each individual paw of the mouse(max=20). The results are expressed as mean±SEM in each group.

The results, as shown in FIG. 4, indicate that an arthritic score wasmeasurable in the C17.15-treated mice only after day 50 post-treatment,and that the peak mean arthritic score obtained with the C17.15-treatedmice was at least 5-fold lower than that measured in the IgG-treatedmice. This demonstrated that the rat anti-mouse IL-12 antibody C17.15prevented the development of collagen-induced arthritis in mice.

B. Suppression of Colitis in Mice by the Rat α-Murine IL-12 AntibodyC17.15

IL-12 has also been demonstrated to play a role in thedevelopment/pathology of colitis. For example, anti-IL-12 antibodieshave been shown to suppress disease in mouse models of colitis, e.g.,TNBS induced colitis IL-2 knockout mice (Simpson et al. (1998) J. Exp.Med. 187(8): 1225-34). Similarly, anti-IL-2 antibodies have beendemonstrated to suppress colitis formation in IL-10 knock-out mice. Theability of the rat anti-mouse IL-12 antibody, C17.15, to suppress TNBScolitis in mice was assessed in two studies (Davidson et al. (1998) J.Immunol. 161(6): 3143-9).

In the first study, colitis was induced in pathogen free SJL mice by theadministration of a 150 μL 50% ethanol solution containing 2.0 mg TNBSdelivered via a pediatric umbilical artery catheter into the rectum.Control animals were treated with a 150 μL 50% ethanol solution only. Asingle dose of 0.75, 0.5, 0.25, or 0.1 mg C17.15 or 0.75 mg control ratIgG2a was given intravenously via the tail vein at day 11, and thetherapeutic effect of the treatment was assessed by weighing the animalson days 11 and 17, and histological scoring at day 17. The weight of themice treated with C17.15 increased within 48 hours of antibody treatmentand normalized on day 6 after treatment. The effect of treatment withC17.15 was confirmed histologically. Further, assessments of IFN-γsecretion by CD4⁺ T-cells from spleen and colon of the treated mice, aswell as IL-12 levels from spleen or colon-derived macrophages from thetreated mice were also made (see Table 12).

In the second study, the dosing was optimized and the mice were treatedwith a total dose of 0.1 mg or 0.5 mg C17.15 or 0.1 mg control IgG2a,respectively, split between days 12 and 14. It was found that theadministration of C17.15 in a single dose at the dosage of 0.1 mg/mouseor 0.25 mg/mouse led to only partial improvement in TNBS-induced colitisand did not result in a significant reduction in the CD4⁺ T cellproduction of IFN-γ in vitro, but did result in a significant decreasein secretion of IL-12, compared to untreated controls. At a single doseof 0.5 mg/mouse or greater a response was observed. Taking the lowestdose of antibody tested and administering it in two divided injections(at days 12 and 14) improved the dosing regimen, indicating thatmultiple low doses can be more effective than a single bolus dose. Thedata obtained are shown in Table 12.

TABLE 12 Anti-mouse Il-12 mAb C17.15 Suppresses Established Colitis inMice IFN-γ IL-12 spleen spleen Disease Weight (g) CD4⁺ macro- InductionTreatment Day Day cells phages Day 0 Day 11 11 17 (U/mL) (pg/ml) TNBS +Ethanol Control IgG2a 16.0 15.26 3326 300 0.75 mg TNBS + Ethanol C17.150.75 mg 16.0 20.21 1732 0 TNBS + Ethanol C17.15 0.5 mg 16.36 19.94 17230 TNBS + Ethanol C17.15 0.25 mg 16.28 17.7 3618 7 TNBS + Ethanol C17.150.1 mg 16.2 17.98 3489 22 Ethanol control — 20.76 21.16 1135 0

Administration of C17.15 monoclonal anti-IL-12 in two divided dosesspaced one day apart totaling 0.1 mg/mouse or 0.05 mg/mouse led tocomplete reversal of colitis as assessed by wasting and macroscopicappearance of the colon. In addition, this dose schedule led tosignificant down-regulation of lamina propria T-cell production of IFN-γand macrophage production of IL-12, so that the latter were comparableto levels seen in control ethanol-treated mice without TNBS-colitis.Thus, C17.15 administration to mouse models for TNBS colitis reversedthe progression of the disease in a dose-dependent manner.

C. Suppression of Experimental Autoimmune Encephalomyelitis (EAE) inMice by α-IL-12 Antibodies

It is commonly believed that IL-12 plays a role in the pathogenesis ofmultiple sclerosis (MS). The inducible IL-12 p40 message has been shownto be expressed in acute plaques of MS patients but not in inflammatorybrain infarct lesions (Windhagen, A. et al. (1995) J. Exp. Med. 182:1985-1996). T cells from MS patients (but not control T cells) stimulateIL-12 production from antigen-presenting cells through unregulated CD40Lexpression (Balashov, K. E. et al. (1997) Proc. Natl. Acad. Sci. USA 94:599-603). MS patients have enhanced IFN-γ secretion that can be blockedwith α-IL-12 antibodies in vitro (Balashov, K. E. et al. (1997) Proc.Natl. Acad. Sci. USA 94: 599-603). Elevated levels of serum IL-12 aredetected in MS patients, but not in other neurological diseases(Nicoletti, F. et al. (1996) J. Neuroimmunol. 70: 87-90). IncreasedIL-12 production has been shown to correlate with disease activity in MSpatients (Cormabella, M. et al. (1998) J. Clin. Invest. 102: 671-678).The role of IL-12 in the pathogenesis of a murine model of multiplesclerosis, experimental autoimmune encephalomyelitis (EAE), has beenstudied (Leonard, J. P. et al. (1995) J. Exp. Med. 181: 281-386;Banerjee, S. et al. (1998) Arthritis Rheum. (1998) 41: S33; and Segal,B. M. et al. (1998) J. Exp. Med. 187: 537-546). The disease in thismodel is known to be induced by T cells of the TH₁ subset. Therefore,the ability of α-IL-12 antibodies to prevent the onset of acute EAE wasassessed.

An α-IL-12 antibody was found to be able to inhibit the onset of acuteEAE, to suppress the disease after onset, and to decrease the severityof relapses in mice immunized with the autoantigen, myelin basic protein(Banerjee, S. et al. (1998) Arthritis Rheum. (1998) 41: S33). Thebeneficial effects of α-IL-12 antibody treatment in the mice persistedfor over two months after stopping treatment. It has also beendemonstrated that anti-IL-12 antibodies suppress the disease in micethat are recipients of encephalitogenic T cells by adoptive transfer(Leonard, J. P. et al. (1995) J. Exp. Med. 181: 281-386).

Example 9 Clinical Pharmacology of J695

In a double blind, crossover study, 64 healthy, human male subjects wereadministered ascending doses of J695 or placebo. Measurement ofcomplement fragment C3a prior to and 0.25 h after dosing did notdemonstrate activation of the complement system. CRP and fibrinogenlevels were only increased in subjects in whom symptoms of concurrentinfections were observed.

All subjects survived and the overall tolerability of J695 was verygood. In no case did treatment have to be stopped because of adverseevents (AEs). The most commonly observed AEs were headache and commoncold/bronchitis, neither of which were categorized as severe.

One of the study subjects, a 33-year-old single male, was suffering frompsoriasis guttata at the start of the study. According to the randomizedstudy design, this subject by chance received 5 mg/kg J695 by SCadministration. Ten days prior to administration of the antibody, thesubject showed only small discrete papular lesions on the arms and legs.At the time of the antibody administration, the subject displayedincreased reddening, thickness of the erythematous plaques, andincreased hyperkaratosis. One week after J695 administration, thesubject reported an improvement in skin condition, including flatteningof the lesions and a decrease in scaling. Shortly after the secondadministration of J695 (5 mg/kg IV), the subject's skin was totallycleared of psoriatic lesions, in the absence of any local treatment.Erythematous plaques covered with white scales reappeared concomitantwith the expected clearance of J695 after the second administration ofantibody.

Example 10 Comparison of J695 Produced by Two CHO Cell Lines

For recombinant expression of J695, a recombinant expression vectorencoding both the antibody heavy chain and the antibody light chain isintroduced into dhfr-CHO cells (Urlaub, G. and Chasin, L. A. (1980)Proc. Natl. Acad. Sci. USA 77:4216-4220) by calcium phosphate-mediatedtransfection. Within the recombinant expression vector, the antibodyheavy and light chain genes are each operatively linked toenhancer/promoter regulatory elements (e.g., derived from SV40, CMV,adenovirus and the like, such as a CMV enhancer/AdMLP promoterregulatory element or an SV40 enhancer/AdMLP promoter regulatoryelement) to drive high levels of transcription of the genes. Therecombinant expression vector also carries a DHFR gene, which allows forselection of CHO cells that have been transfected with the vector usingmethotrexate selection/amplification.

One hundred and fifty micrograms of an expression vector encoding thepeptide sequences of the human antibody J695 were dissolved in 2.7 mlwater in a 50 ml conical tube. Three hundred μL of 2.5 M CaCl₂ wereadded and this DNA mixture was added dropwise to 3 ml of 2×HEPESbuffered saline in a 50 ml conical tube. After vortexing for 5 sec andincubating at room temperature for 20 min, 1 mL was distributed evenlyover each plate (still in F12 medium), and the plates were incubated at37° C. for 4 h. Liquid was removed by aspiration and 2 ml of 10% DMSO inF12 were added to each plate. The DMSO shock continued for 1 min, afterwhich the DMSO was diluted by the addition of 5 ml PBS to each plate.Plates were washed twice in PBS, followed by the addition of 10 ml ofalpha MEM, supplemented with H/T and 5% FBS (selective for cellsexpressing DHFR) and overnight incubation at 37° C. Cells were seededinto 96-well plates at a density of 100 cells per well, and plates wereincubated at 37° C., 5% CO₂ for two weeks, with one change of medium perweek.

Five days after the final medium change, culture supernatants werediluted 1:50 and tested using an ELISA specific for human IgG gammachain. The clones yielding the highest ELISA signal were transferredfrom the 96-well plates to 12-well plates in 1.5 ml/well of Alpha MEM+5%dialyzed serum. After 3 days, another ELISA specific for human IgG gammachain was performed, and the 12 clones with the greatest activity weresplit into the alpha MEM+5% dialyzed serum and 20 nM MTX. Cell line031898 218 grew in the presence of 20 nM MTX without any apparent celldeath or reduction in growth rate, produced 1.8 μg/ml hIgG in athree-day assay. T-25 cultures of 031898 218, growing in mediumcontaining MTX, produced an average of 11.9 μg/ml of J695. The line,designated ALP903, was adapted to growth in suspension under serum-freeconditions, where it produced 7.5 pg J695/cell/24 h.

ALP903 cells, after initial selection in alpha MEM/5% FBS/20 nM MTXmedium, were passed again in 20 nM MTX. The cells were cultured under100 nM MTX selection, followed by passaging in 500 nM MTX twice in thenext 30 days. At that time the culture was producing 32 μg J695/mL/24 h.The culture was subcloned by limiting dilution. Subclone 218-22 produced16.5 μg/mL in a 96-well plate in 2 days and 50.3 μg/mL of J695 in a12-well dish in 2 days. Clone 218-22 was cultured in alpha MEM/5%dialyzed FBS/500 nM MTX for 38 days, followed by adaptation toserum-free spinner culture, as above. The average cell-specificproductivity of the serum-free suspension culture, designated ALP 905,was 58 pg/cell/24 h.

The first cell line used to produce J695 (ALP 903) resulted in loweryields of the antibody from culture than a second cell line, ALP 905. Toassure that the ALP 905-produced J695 was functionally identical to thatproduced from ALP 903, both batches of antibodies were assessed forIL-12 affinity, for the ability to block IL-12 binding to cellularreceptors, for the ability to inhibit IFN-γ induction by IL-12, and forthe ability to inhibit IL-12-mediated PHA blast proliferation.

The affinities of J695 batches ALP 903 and ALP 905 for IL-12 weredetermined by measuring the kinetic rate constants of binding to IL-12by surface plasmon resonance studies (BIAcore analyses). The off-rateconstant (k_(d)) and the on-rate constant (k_(a)) of antibody batchesALP903 and ALP905 for binding to rhIL-12 were determined in threeexperiments (as described in Example 3). The affinity, K_(d), of bindingto IL-12 was calculated by dividing the off-rate constant by the on-rateconstant. K_(d) was calculated for each separate experiment and thenaveraged. The results showed that the determined kinetic parameters andaffinity of binding to rhIL-12 were very similar for J695 batches ALP903 and ALP 905: the calculated K_(d) was 1.19±0.22×10⁻¹⁰ M for batchALP 903 and 1.49±0.47×10⁻¹⁰ M for batch ALP 905 (see Table 13).

The ability of J695 derived from both ALP 903 and ALP 905 to blockbinding of rhIL-12 to IL-12 receptors on human PHA-activatedT-lymphoblasts was assessed (see Example 3). Each sample of J695 wastested at a starting concentration of 1×10⁻⁸ with 10-fold serialdilutions. The antibody was preincubated for 1 hour at 37° C. with 50 pM[¹²⁵I]-human IL-12 in binding buffer. PHA blast cells were added to theantibody/[¹²⁵I]-human IL-12 mixture and incubated for 2 h at roomtemperature. Cell bound radioactivity was separated from free[¹²⁵I]-IL-12 by centrifugation and washing steps, and % inhibition wascalculated. The IC₅₀ values for J695 were determined from the inhibitioncurves using 4-parameter curve fitting and were confirmed by twoindependent experiments. Incubations were carried out in duplicate. Theresults for the two batches of J695 were very similar (see Table 13).

The ability of J695 from both ALP 903 and ALP 905 cells to inhibitrhIL-12-induced IFN-γ production by human PHA-activated lymphoblasts invitro was assessed. Serial dilutions of J695 were preincubated with 200pg/mL rhIL-12 for 1 h at 37° C. PHA lymphoblast cells were added andincubated for 18 hours at 37° C. After incubation, cell free supernatantwas withdrawn and the level of human IFN-γ determined by ELISA. The IC₅₀values from the inhibition curves were plotted against the antibodyconcentration using 4-parameter curve fitting. The results demonstratethat the ability of the two batches to inhibit IFN-γ production is verysimilar.

The in vitro PHA blast cell proliferation assay was used to measure theneutralization capacity of ALP 903 and ALP 905 J695 for rhIL-12. Serialdilutions of J695 of each type were preincubated with 230 pg/mL humanIL-12 for 1 h at 37° C. Next PHA blast cells were added and incubatedfor 3 days at 37° C. The cells were then labeled for 6 hours with 1γCi/well [³H]-thymidine. The cultures were harvested and [³H]-thymidineincorporation measured. Non-specific proliferation (background) wasmeasured in the absence of rhIL-12. The IC₅₀ values for ALP 903 and ALP905 J695 were found to be very similar and are set forth in Table 13.

The activity of the J695 antibodies in neutralizing rhIL-12 activity, inblocking IL-12 binding to cell surface receptors, and in binding torhIL-12 did not significantly differ from batch ALP 903 to batch ALP905, and thus the antibodies produced from these two different celltypes were equivalent.

TABLE 13 Comparison of the Properties of J695 lots ALP 903 and ALP 905PHA blast IFN-γ k_(a), On-rate k_(d), Off-rate RB assay Assay IC₅₀ AssayIC₅₀ Antibody (M⁻¹, s⁻¹) (s⁻¹) K_(d) (M) IC₅₀ (M) (M) (M) J695 3.75 ×10⁵ 4.46 × 10⁻⁵ 1.19 × 10⁻¹⁰ 3.4 × 10⁻¹¹ 5.5 × 10⁻¹² 5.8 × 10⁻¹² ALP 903J695 3.91 × 10⁵ 5.59 × 10⁻⁵ 1.49 × 10⁻¹⁰ 3.0 × 10⁻¹¹ 4.4 × 10⁻¹² 4.3 ×10⁻¹² ALP 905

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of treating a human subject suffering from an autoimmunedisorder, comprising administering to the human subject an effectiveamount of a human antibody, or antigen-binding portion thereof, whichbinds to an epitope of the p40 subunit of IL-12, thereby treating saidsubject.
 2. The method of claim 1, wherein the autoimmune disorder ispsoriasis.
 3. The method of claim 1, wherein the autoimmune disorder isrheumatoid arthritis.
 4. The method of claim 1, wherein the autoimmunedisorder is Crohn's disease.
 5. The method of claim 1, wherein theautoimmune disorder is Multiple Sclerosis.
 6. The method of claim 1,wherein the antibody, or antigen-binding portion thereof, binds to theepitope of the p40 subunit when the p40 subunit is bound to the p35subunit of IL-12.
 7. The method of claim 1, wherein the antibody, orantigen-binding portion thereof, binds to the epitope of the p40 subunitwhen the p40 subunit is bound to a p19 subunit.
 8. The method of claim1, wherein the antibody, or antigen-binding portion thereof, binds tothe epitope of the p40 subunit when the p40 subunit is bound to the p35subunit of IL-12 and when the p40 subunit is bound to a p19 subunit. 9.The method of claim 1, wherein the antibody, or antigen binding portionthereof, binds to an epitope of the p40 subunit of IL-12 to which anantibody selected from the group consisting of Y61 and J695 binds. 10.The method of claim 1, wherein the antibody further binds to a firstheterodimer and also binds to a second heterodimer, wherein the firstheterodimer comprises the p40 subunit of IL-12 and the p35 subunit ofIL-12, and wherein the second heterodimer comprises the p40 subunit ofIL-12 and a p19 subunit.
 11. The method of claim 10, wherein theantibody neutralizes the biological activity of the first heterodimer.12. The method of claim 10, wherein the antibody neutralizes thebiological activity of the second heterodimer.
 13. The method of claim10, wherein the antibody neutralizes the biological activity of thefirst heterodimer and the second heterodimer.
 14. The method of claim10, wherein the antibody, or antigen binding portion thereof, inhibitsphytohemagglutinin blast proliferation in an in vitro PHA assay with anIC₅₀ of 1×10⁻⁹ M or less, or which inhibits human IFNγ production withan IC₅₀ of 1.6×10⁻¹⁰ M or less.
 15. The method of claim 1, wherein theantibody, or antigen binding portion thereof, dissociates from the p40subunit of IL-12 with a K_(d) of 1.34×10⁻¹⁰ M or less or a k_(off) rateconstant of 1×10⁻³ s⁻¹ or less, as determined by surface plasmonresonance.
 16. The method of claim 1, wherein the antibody, or antigenbinding portion thereof, comprises a heavy chain CDR3 comprising theamino acid sequence of SEQ ID NO: 25 and a light chain CDR3 comprisingthe amino acid sequence of SEQ ID NO: 26;
 17. The method of claim 16,wherein the antibody, or antigen binding portion thereof, comprises aheavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27 anda light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 28.18. The method of claim 16, wherein the antibody, or antigen bindingportion thereof, comprises a heavy chain CDR1 comprising the amino acidsequence of SEQ ID NO: 29 and a light chain CDR1 comprising the aminoacid sequence of SEQ ID NO:
 30. 19. A method of treating a human subjectsuffering from an autoimmune disorder, comprising administering to thehuman subject an effective amount of a human antibody, orantigen-binding portion thereof, which binds to an interleukincomprising a p40 subunit, thereby treating said subject.
 20. The methodof claim 19, wherein the autoimmune disorder is psoriasis.
 21. Themethod of claim 19, wherein the autoimmune disorder is rheumatoidarthritis.
 22. The method of claim 19, wherein the autoimmune disorderis Crohn's disease.
 23. The method of claim 19, wherein the autoimmunedisorder is Multiple Sclerosis.
 24. The method of claim 19, wherein theinterleukin comprises a p40 subunit and a p35 subunit.
 25. The method ofclaim 24, wherein the interleukin is IL-12.
 26. The method of claim 19,wherein the interleukin comprises a p40 subunit and a p19 subunit. 27.The method of claim 19, wherein the antibody, or antigen binding portionthereof, binds to an epitope of the p40 subunit.
 28. The method of claim19, wherein the antibody, or antigen binding portion thereof, binds toan epitope of the p40 subunit to which an antibody selected from thegroup consisting of Y61 and J695 binds.
 29. The method of claim 19,wherein the antibody, or antigen binding portion thereof, dissociatesfrom the p40 subunit of the interleukin with a K_(d) of 1.34×10⁻¹⁰ M orless or a k_(off) rate constant of 1×10⁻³s⁻¹ or less, as determined bysurface plasmon resonance.
 30. The method of claim 19, wherein theantibody, or antigen binding portion thereof, neutralizes the biologicalactivity of the interleukin.
 31. The method of claim 30, wherein theantibody, or antigen binding portion thereof, inhibitsphytohemagglutinin blast proliferation in an in vitro PHA assay with anIC₅₀ of 1×10⁻⁹ M or less, or which inhibits human IFNγ production withan IC₅₀ of 1.6×10⁻¹⁰ M or less.
 32. The method of claim 19, wherein theantibody, or antigen binding portion thereof, comprises a heavy chainCDR3 comprising the amino acid sequence of SEQ ID NO: 25 and a lightchain CDR3 comprising the amino acid sequence of SEQ ID NO:
 26. 33. Themethod of claim 32, wherein the antibody, or antigen binding portionthereof, comprises a heavy chain CDR2 comprising the amino acid sequenceof SEQ ID NO: 27 and a light chain CDR2 comprising the amino acidsequence of SEQ ID NO:
 28. 34. The method of claim 32, wherein theantibody, or antigen binding portion thereof, comprises a heavy chainCDR1 comprising the amino acid sequence of SEQ ID NO: 29 and a lightchain CDR1 comprising the amino acid sequence of SEQ ID NO:
 30. 35. Amethod of treating a human subject suffering from psoriasis, comprisingadministering to the human subject an effective amount of a humanantibody, or antigen-binding portion thereof, which binds to an epitopeof the p40 subunit of IL-12, thereby treating the subject.
 36. Themethod of claim 35, wherein the subject exhibits an improvement in skincondition for an extended period following administration of theantibody, or antigen-binding portion thereof.
 37. The method of claim36, wherein the subject exhibits flattening of plaques.
 38. The methodof claim 36, wherein the subject exhibits a decrease in scaling.
 39. Themethod of claim 35, wherein the subject exhibits a total clearance ofplaques for an extended period following administration of the antibody,or antigen-binding portion thereof.
 40. The method of claim 35, whereinsaid effective amount is about 0.01 mg/kg to about 10 mg/kg.
 41. Themethod of claim 35, wherein said antibody, or antigen-binding portionthereof, is administered to said subject subcutaneously.
 42. The methodof claim 35, wherein said antibody, or antigen-binding portion thereof,is administered to said subject intravenously.
 43. The method of claim35, wherein the antibody, or antigen-binding portion thereof, binds tothe epitope of the p40 subunit when the p40 subunit is bound to the p35subunit of IL-12.
 44. The method of claim 35, wherein the antibody, orantigen-binding portion thereof, binds to the epitope of the p40 subunitwhen the p40 subunit is bound to a p19 subunit.
 45. The method of claim35, wherein the antibody, or antigen-binding portion thereof, binds tothe epitope of the p40 subunit when the p40 subunit is bound to the p35subunit of IL-12 and when the p40 subunit is bound to a p19 subunit. 46.The method of claim 35, wherein the antibody, or antigen binding portionthereof, binds to an epitope of the p40 subunit of IL-12 to which anantibody selected from the group consisting of Y61 and J695 binds. 47.The method of claim 35, wherein the antibody further binds to a firstheterodimer and also binds to a second heterodimer, wherein the firstheterodimer comprises the p40 subunit of IL-12 and the p35 subunit ofIL-12, and wherein the second heterodimer comprises the p40 subunit ofIL-12 and a p19 subunit.
 48. The method of claim 47, wherein theantibody neutralizes the biological activity of the first heterodimer.49. The method of claim 47, wherein the antibody neutralizes thebiological activity of the second heterodimer.
 50. The method of claim47, wherein the antibody neutralizes the biological activity of thefirst heterodimer and the second heterodimer.
 51. The method of claim47, wherein the antibody, or antigen binding portion thereof, inhibitsphytohemagglutinin blast proliferation in an in vitro PHA assay with anIC₅₀ of 1×10⁻⁹ M or less, or which inhibits human IFNγ production withan IC₅₀ of 1.6×10⁻¹⁰ M or less.
 52. The method of claim 35, wherein theantibody, or antigen binding portion thereof, dissociates from the p40subunit of IL-12 with a K_(d) of 1.34×10⁻¹⁰ M or less or a k_(off) rateconstant of 1×10⁻³s⁻¹ or less, as determined by surface plasmonresonance.
 53. The method of claim 35, wherein the antibody, or antigenbinding portion thereof, comprises a heavy chain CDR3 comprising theamino acid sequence of SEQ ID NO: 25 and a light chain CDR3 comprisingthe amino acid sequence of SEQ ID NO:
 26. 54. The method of claim 53,wherein the antibody, or antigen binding portion thereof, comprises aheavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27 anda light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 28.55. The method of claim 53, wherein the antibody, or antigen bindingportion thereof, comprises a heavy chain CDR1 comprising the amino acidsequence of SEQ ID NO: 29 and a light chain CDR1 comprising the aminoacid sequence of SEQ ID NO:
 30. 56. A method for inhibiting the activityof an interleukin comprising a p40 subunit in a human subject sufferingfrom psoriasis, comprising administering to the human subject aneffective amount of a human antibody, or antigen-binding portionthereof, which to an interleukin comprising a p40 subunit, such that thepsoriasis is treated.
 57. The method of claim 56, wherein the subjectexhibits an improvement in skin condition for an extended periodfollowing administration of the antibody, or antigen-binding portionthereof.
 58. The method of claim 57, wherein the subject exhibitsflattening of plaques.
 59. The method of claim 57, wherein the subjectexhibits a decrease in scaling.
 60. The method of claim 56, wherein thesubject exhibits a total clearance of plaques for an extended periodfollowing administration of the antibody, or antigen-binding portionthereof.
 61. The method of claim 56, wherein said effective amount isabout 0.01 mg/kg to about 10 mg/kg.
 62. The method of claim 56, whereinsaid antibody, or antigen-binding portion thereof, is administered tosaid subject subcutaneously.
 63. The method of claim 56, wherein saidantibody, or antigen-binding portion thereof, is administered to saidsubject intravenously.
 64. The method of claim 56, wherein theinterleukin comprises a p40 subunit and a p35 subunit.
 65. The method ofclaim 64, wherein the interleukin is IL-12.
 66. The method of claim 56,wherein the interleukin comprises a p40 subunit and a p19 subunit. 67.The method of claim 56, wherein the antibody, or antigen binding portionthereof, binds to an epitope of the p40 subunit.
 68. The method of claim56, wherein the antibody, or antigen binding portion thereof, binds toan epitope of the p40 subunit to which an antibody selected from thegroup consisting of Y61 and J695 binds.
 69. The method of claim 56,wherein the antibody, or antigen binding portion thereof, dissociatesfrom the p40 subunit of the interleukin with a K_(d) of 1.34×10⁻¹⁰ M orless or a k_(off) rate constant of 1×10⁻³s⁻¹ or less, as determined bysurface plasmon resonance.
 70. The method of claim 56, wherein theantibody, or antigen binding portion thereof, neutralizes the biologicalactivity of the interleukin.
 71. The method of claim 70, wherein theantibody, or antigen binding portion thereof, inhibitsphytohemagglutinin blast proliferation in an in vitro PHA assay with anIC₅₀ of 1×10⁻⁹ M or less, or which inhibits human IFNγ production withan IC₅₀ of 1.6×10⁻¹⁰ M or less.
 72. The method of claim 56, wherein theantibody, or antigen binding portion thereof, comprises a heavy chainCDR3 comprising the amino acid sequence of SEQ ID NO: 25 and a lightchain CDR3 comprising the amino acid sequence of SEQ ID NO: 26;
 73. Themethod of claim 72, wherein the antibody, or antigen binding portionthereof, comprises a heavy chain CDR2 comprising the amino acid sequenceof SEQ ID NO: 27 and a light chain CDR2 comprising the amino acidsequence of SEQ ID NO:
 28. 74. The method of claim 72, wherein theantibody, or antigen binding portion thereof, comprises a heavy chainCDR1 comprising the amino acid sequence of SEQ ID NO: 29 and a lightchain CDR1 comprising the amino acid sequence of SEQ ID NO:
 30. 75. Amethod of treating a human subject suffering from psoriasis, comprisingadministering to the human subject about 0.01 mg/kg to about 10 mg/kg ofa human antibody, or antigen-binding portion thereof, which binds to anepitope of the p40 subunit of IL-12, thereby treating said subject. 76.The method of claim 75, wherein said antibody, or antigen-bindingportion thereof, is administered to said subject subcutaneously.
 77. Themethod of claim 75, wherein said antibody, or antigen-binding portionthereof, is administered to said subject intravenously.
 78. The methodof claim 75, wherein said antibody, or antigen-binding portion thereof,is administered in an amount of about 1 mg/kg to about 10 mg/kg.
 79. Themethod of claim 75, wherein said antibody, or antigen-binding portionthereof, is administered using a formulation further comprising at leastone additional agent selected from the group consisting of a buffer, apolyalcohol and a surfactant.
 80. The method of claim 79, wherein saidbuffer is selected from the group consisting of L-histidine, sodiumsuccinate, sodium citrate, sodium phosphate and potassium phosphate. 81.The method of claim 79, wherein said polyalcohol is selected from thegroup consisting of mannitol and sorbitol.
 82. The method of claim 79,wherein said surfactant is selected from the group consisting ofpolysorbate 80, polysorbate 20 and BRIJ surfactants.
 83. The method ofclaim 79, wherein said formulation further comprises methionine.
 84. Themethod of claim 79, wherein said formulation comprises a polyalcohol, asurfactant, and a buffer comprising L-histidine.
 85. The method of claim84, wherein the polyalcohol is mannitol.
 86. The method of claim 84,wherein the surfactant is polysorbate
 80. 87. The method of claim 84,wherein said formulation further comprises methionine.
 88. The method ofclaim 87, wherein said composition comprises (a) 1-10% mannitol, (b)0-0.05% polysorbate-80, (c) 1-50 mM methionine, and (d) a buffercomprising 1-50 mM L-histidine, with a pH of 5 to
 7. 89. The method ofclaim 87, wherein said composition comprises (a) 2-4% mannitol, (b)0.005-0.01% polysorbate-80, (c) 5-10 mM methionine, and (d) a buffercomprising 5-10 mM L-histidine, with a pH of 5 to
 7. 90. A method oftreating a subject suffering from psoriasis, comprising subcutaneouslyadministering to the subject a human antibody, or antigen-bindingportion thereof, which binds to an epitope of the p40 subunit of IL-12,wherein said antibody, or antigen-binding portion thereof, isadministered using a formulation comprising: (a) about 1-10 mg/kg ofsaid antibody, or antigen-binding portion thereof, (b) 2-4% mannitol,(c) 0.005-0.01% polysorbate-80, (d) 5-10 mM methionine, and (e) a buffercomprising 5-10 mM L-histidine, with a pH of 5 to 7, thereby treatingsaid subject.